Background: Mammals synthesize endogenous morphine; the enzyme catalyzing thebaine O 6 -demethylation, a key late step, is uncharacterized. Results: Human cytochromes P450 (P450) 3A4 and 3A5 catalyzed thebaine O 6 -demethylation; the P450 3A-selective drug ketoconazole inhibited the reaction in human liver microsomes and rat brain homogenates. Conclusion: P450s 3A4 and 3A5 catalyze thebaine O 6 -demethylation in humans. Significance: Enzymes catalyzing all oxidations in the latter steps of mammalian morphine biosynthesis have been identified.