We have investigated cytochrome P450 (P450) and associated monooxygenase activities in human brain mitochondria isolated from eight regions of four human brain samples obtained at autopsy. P450-associated monooxygenase activities including aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD), p-nitrophenol hydroxylase (PNPH), and N-nitrosodimethylamine N-demethylase (ND-MAD) were detectable in the mitochondria from human brain regions. Immunoblot experiments using antisera to purified rat liver microsomal P450, namely P4502B1/2, P4501A1/2, and P4502E1, revealed immunoreactive bands in isolated mitochondria from different regions of the human brain. The antibody to P4502B1/2 and P4501A1/2 inhibited the human brain mitochondrial APD and ECD activities, respectively. The addition of antiserum to microsomal NADPH cytochrome P450 reductase did not affect the mitochondrial P450-associated monooxygenase activities, although it completely inhibited the corresponding activities in brain microsomes. Overall, the present study demonstrates, in human brain mitochondria, the presence of multiple forms of P450 belonging to the 1A, 2B, and 2E subfamilies that are involved in xenobiotic metabolism.