The inhibition of rat liver microsomal cytochrome P-450 by benzyl hydrodisulfide has been examined as a model system for the inactivation of cytochrome P-450 seen during the microsomal metabolism of thiono-sulfur-containing compounds. Benzyl hydrodisulfide decreased enzymatic activity toward benzphetamine when incubated with hepatic microsomes prior to the assay for monooxygenase activity. In addition, incubation of microsomes with the hydrodisulfide caused a decrease in the level of cytochrome P-450 detectable as its carbon monoxide complex as well as a decrease in heme detectable as its pyridine-hemochromogen. In a typical experiment, the loss of enzymatic activity, cytochrome P-450, and heme were 65, 56, and 51%, respectively. These data suggest that the major loss of monooxygenase activity and of cytochrome P-450 which was seen on incubation of microsomes with benzyl hydrodisulfide results from an alteration in the structure of the heme moiety of cytochrome P-450. A similar alteration in the heme moiety of cytochrome P-450 is believed to be responsible, in part, for the loss of monooxygenase activity and cytochrome P-450 seen on incubation of parathion and other thiono-sulfur-containing compounds with hepatic microsomes or a reconstituted cytochrome P-450-containing monooxygenase system.