The metabolism of (methylenedioxy)amphetamine (MDA) and (methylenedioxy)methamphetamine (MDMA) was examined in microsomal preparations from rat brains. The products generated from MDA and MDMA were identified as dihydroxyamphetamine (DHA) and dihydroxymethamphetamine (DHMA), respectively. The demethylenation reaction required NADPH and was strongly inhibited by CO/O2 (4:1 v/v), suggesting that the formation of DHA and DHMA is mediated by cytochrome P450. The conversion was inhibited by desipramine, imipramine, and methimazole, whereas SKF-525A and alpha-naphthoflavone had little effect. Lineweaver-Burk plots of MDA and MDMA demethylenation were biphasic in both cases, indicating that multiple isozymes may participate in the oxidation. The microsomal preparation showed no significant stereoselectivity in the demethylenation of either MDA or MDMA. Catechol formation differed with the incubation buffer and was 2.6 times greater when phosphate rather than HEPES buffer was used. This difference disappeared, however, when desferrioxamine B methanesulfonate (desferal) and hydroxyl radical (.OH) scavenging agents were added to either buffer. The demethylenation was also sensitive to catalase and was stimulated by the addition of ferric ion and EDTA to the microsomal incubation mixture. These results indicate that the demethylenation of MDA and MDMA by rat brain microsomes has a cytochrome P450-mediated component as well as a chemical component involving .OH.