Sections of B. caapi vine are macerated and boiled alone or with leaves from any of a number of other plants, including Psychotria viridis (chacruna) or Diplopterys cabrerana (also known as chaliponga). The resulting brew contains the powerful hallucinogenic alkaloid N,N-dimethyltryptamine (DMT), and MAO inhibiting harmala alkaloids, which are necessary to make the DMT orally active. Though B. caapi is a central ingredient in traditional ayahuasca brews, harmala-containing plants from other plant-medicine cultures, such as Syrian Rue, can be used instead of the vine to make an ayahuasca analogue, yet it isn't considered ayahuasca, as Caapi vine is considered the main plant in the brew.[citation needed]

Brews can also be made with no DMT-containing plants; Psychotria viridis being substituted by plants such as Justicia pectoralis, Brugmansia, or sacred tobacco, also known as Mapacho (Nicotiana rustica), or sometimes left out with no replacement. The potency of this brew varies radically from one batch to the next, both in potency and psychoactive effect, based mainly on the skill of the shaman or brewer, as well as other admixtures sometimes added and the intent of the ceremony.[citation needed] Natural variations in plant alkaloid content and profiles also affect the final concentration of alkaloids in the brew, and the physical act of cooking may also serve to modify the alkaloid profile of harmala alkaloids.

Individual polymorphisms in the cytochrome P450-2D6 enzyme affect the ability of individuals to metabolize harmine. Some natural tolerance to habitual use of ayahuasca (roughly once weekly) may develop through upregulation of the serotonergic system. A phase 1 pharmacokinetic study on Ayahuasca (as Hoasca) with 15 volunteers was conducted in 1993, during the Hoasca Project. A review of the Hoasca Project has been published.

Harmine compounds are of beta-carboline origin. The three most studied beta-carboline compounds found in the B. caapi vine are harmine, harmaline and tetrahydroharmine. Harmine and harmaline are selective and reversible inhibitors of MAO-A, while tetrahydroharmine is a weak serotonin uptake inhibitor. This inhibition of MAO-A allows DMT to diffuse unmetabolized past the membranes in the stomach and small intestine and eventually get through the blood-brain barrier to activate receptor sites in the brain. Without RIMAs or the MAOI of MAO-A, DMT would be metabolized in the digestive tract and would not have an effect when taken orally.

Ayahuasca is used largely as a religious sacrament. Those whose usage of ayahuasca is performed in non-traditional contexts often align themselves with the philosophies and cosmologies associated with ayahuasca shamanism, as practiced among indigenous peoples like the Urarina of Peruvian Amazonia. The religion Santo Daime uses it.

While non-native users know of the spiritual applications of ayahuasca, a less well-known traditional usage[specify] focuses on the medicinal properties of ayahuasca. Its purgative properties are highly important (many refer to it as la purga, "the purge"). The intense vomiting and occasional diarrhea it induces can clear the body of worms and other tropical parasites, and harmala alkaloids themselves have been shown to be anthelmintic Thus, this action is twofold; a direct action on the parasites by these harmala alkaloids (particularly harmine in ayahuasca) works to kill the parasites, and parasites are expelled through the increased intestinal motility that is caused by these alkaloids.

Dietary taboos are often associated with the use of Ayahuasca. In the rainforest, these tend towards the purification of one's self - abstaining from spicy and heavily-seasoned foods, excess fat, salt, caffeine, acidic foods (such as citrus) and sex before, after, or both before and after a ceremony. A diet low in foods containing tyramine has been recommended, as the speculative interaction of tyramine and MAOIs could lead to a hypertensive crisis. However, evidence indicates that harmala alkaloids act only on MAO-A, in a reversible way similar to moclobemide (an antidepressant that does not require dietary restrictions). Psychonautic experiments and the absence of dietary restrictions in the highly urban Brazilian ayahuasca church União do Vegetal also suggest that the risk is much lower than perceived, and probably non-existent.

The name 'ayahuasca' specifically refers to a botanical decoctions that contains Banisteriopsis caapi. A synthetic version, known as pharmahuasca is a combination of an appropriate MAOI and typically DMT. In this usage, the DMT is generally considered the main psychoactive active ingredient, while the MAOI merely preserves the psychoactivity of orally ingested DMT, which would otherwise be destroyed in the gut before it could be absorbed in the body. Thus, ayahuasqueros and most others working with the brew maintain that the B. caapi vine is the defining ingredient, and that this beverage is not ayahuasca unless B. caapi is in the brew. The vine is considered to be the "spirit" of ayahuasca, the gatekeeper and guide to the otherworldly realms.

In some areas[specify], it is even said that the chakruna or chaliponga admixtures are added only to make the brew taste sweeter. This is a strong indicator of the often wildly divergent intentions and cultural differences between the native ayahuasca-using cultures and psychedelics enthusiasts in other countries.

In modern Europe and North America, ayahuasca analogues are often prepared using non-traditional plants which contain the same alkaloids. For example, seeds of the Syrian rue plant can be used as a substitute for the ayahuasca vine, and the DMT-rich Mimosa hostilis is used in place of chakruna. Australia has several indigenous plants which are popular among modern ayahuasqueros there, such as various DMT-rich species of Acacia.