The antidepressants, imipramine and mianserin, have been reported to cause liver damage. We investigated a role of cytochrome P450 (CYP)-mediated formation of a reactive metabolite in antidepressant-induced acute cell injury using hepatocytes isolated from male and female Wistar rats, and male Dark Agouti rats, which have different relative abundance of CYP enzymes. Culture of the hepatocytes with imipramine and mianserin caused a marked decrease in glutathione followed by protein thiol, which preceded lactate dehydrogenase leakage. The decreases in glutathione and protein thiol contents by imipramine were significantly slower in hepatocytes from male Dark Agouti rats than those from male Wistar rats, whereas no significant sex difference in Wistar rats was observed. The decrease in thiol by mianserin was significantly slower in hepatocytes from female Wistar than those from male Wistar rats, whereas no significant differences were found between Wistar and Dark Agouti males. Results consistent with alteration of the thiols were obtained for lactate dehydrogenase leakage induced by imipramine and mianserin. These findings indicated that CYP-mediated metabolic activation was involved in acute cell injury induced by the antidepressants; namely a CYP2D enzyme(s), which is deficient in Dark Agouti rats, and a male specific CYP enzyme(s) were suggested to be responsible for the cytotoxicity of imipramine and mianserin, respectively.