The incubation of methimazole (1-methyl-2-thioimidazole, MMI) with rat hepatic microsomes led to the formation of 3-methyl-2-thiohydantoin and N-methylimidazole. An NADPH-stimulated binding of 14C and 35S from [14C]- and [35S]MMI to microsomal macromolecules was also seen. The NADPH-stimulated N-methylimidazole formation and binding of radioactivity from [14C]- and [35S]MMI to microsomal macromolecules appeared to be catalyzed largely by the cytochrome P-450 monoxygenase systems of rat hepatic microsomes. A portion of the radioactivity bound to microsomes incubated with [14C]- and [35S]MMI was released as unchanged MMI on prolonged incubation under acid condition, suggesting that strong binding of MMI to microsomes occurred. A portion of the 35S bound to microsomes incubated with [35S]MMI was released as 35SCN- on incubation of the 35S-labeled microsomes with CN-. A portion of the S released in the metabolism of MMI to N-methylimidazole is in the form of atomic S, which binds to cysteine sulfhydryl groups (R-S-H) in microsomal proteins to form a hydrodisulfide (R-S-S-H). [These results have relevance to the hepatotoxicity of this drug.].