As a selective β1-receptor antagonist, metoprolol tartrate (MTA) is commonly used to treat cardiovascular diseases such as hypertension and angina pectoris. There have been cases of liver injury induced by MTA, but the mechanism of hepatotoxicity induced by MTA is not clear. The purposes of this study were to identify the reactive metabolites of MTA, to determine the pathway for the metabolic activation of MTA, and to define a possible correlation between the metabolic activation and cytotoxicity of MTA. Three oxidative metabolites (M1-M3), a glutathione (GSH) conjugate (M4), and an N-acetyl cysteine (NAC) conjugate (M5) were detected in rat liver microsomal incubations containing MTA and GSH or NAC. M4 was also detected in cultured rat primary hepatocytes and bile of rats given MTA, and M5 was detected in the urine of MTA-treated rats. A quinone methide intermediate may be produced from the metabolic activation process in vitro and in vivo. The metabolite was reactive to glutathione and N-acetyl cysteine. MTA induced marked cytotoxicity in cultured rat primary hepatocytes. Pretreatment of aminobenzotriazole, a nonselective P450 enzyme inhibitor, attenuated the susceptibility of hepatocytes to MTA cytotoxicity.