S-Allylmercaptocysteine (SAMC), one of the water-soluble organosulfur compounds in ethanol extracts of garlic (Allium sativum L.), has been shown to protect mice against acetaminophen (APAP)-induced liver injury. In this study, we examined the mechanisms underlying this hepatoprotection. SAMC (100 mg/kg, p.o.) given 2 and 24 hr before APAP administration (500 mg/kg, p.o.) suppressed the plasma alanine aminotransferase activity increases 3 to 12 hr after APAP administration significantly. The hepatic reduced glutathione levels of vehicle-pretreated mice decreased 1 to 6 hr after APAP administration, but SAMC pretreatment suppressed the reductions 1 to 6 hr after APAP administration significantly. These inhibitory effects of SAMC were dose-dependent (50-200 mg/kg) 6 hr after APAP administration. As SAMC pretreatment (50-200 mg/kg) suppressed hepatic cytochrome P450 2E1-dependent N-nitrosodimethylamine demethylase activity significantly in a dose-dependent manner, we suggest that one of its protective mechanisms is inhibition of cytochrome P450 2E1 activity. SAMC pretreatment also suppressed the increase in hepatic lipid peroxidation and the decrease in hepatic reduced coenzyme Q9 (CoQ9H2) levels 6 hr after APAP administration. The hepatic CoQ9H2 content of the SAMC pretreatment group was maintained at the normal level. Therefore, we suggest that another hepatoprotective mechanism of SAMC may be attributable to its antioxidant activity.