Anandamide (AN) is an arachidonic acid congener, found in the brain, that binds to the cannabinoid receptor and elicits cannabinoid-like pharmacological activity. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to a wide variety of metabolites, yielding many physiologically potent compounds. To determine if AN could be similarly oxidized by P450s, its metabolism by mouse liver and brain microsomes was examined. Mouse hepatic microsomal incubation of AN with NADPH resulted in the generation of at least 20 metabolites, determined after HPLC separation by increased UV-absorbance at 205 nm. Pretreatment of mice with various P450 inducers resulted in increased hepatic microsomal formation of several AN metabolites, with dexamethasone being the most effective inducer. Phenobarbital pretreatment resulted in a metabolic profile similar to that observed after dexamethasone pretreatment, whereas 3-methylcholanthrene pretreatment selectively increased the formation of several other metabolites. Clofibrate pretreatment had no effect on hepatic AN metabolism. Polyclonal antibodies prepared against mouse hepatic P450 3A inhibited the formation of several AN metabolites by hepatic microsomes from untreated mice as well as the formation of those metabolites found to be increased after dexamethasone pretreatment. AN metabolism by brain microsomes resulted in the formation of two NADPH- and protein-dependent metabolites. Hepatic P450 3A antibody partially inhibited the formation of only one of these metabolites. Thus, P450 3A is a major contributor to AN metabolism in the liver but not in the brain. The physiological consequences of P450-mediated AN metabolism remain to be determined.