Temporal variation in metabolism and hepatotoxicity of acetaminophen (APAP) was examined using male ICR mice. Animals were injected with a single dose of APAP (400 mg/kg, i.p.) at 08:00, 14:00 or 20:00 h. APAP at this dose was markedly hepatotoxic to mice when administered at 20:00 h as determined by increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and by decreases in hepatic glucose-6-phosphatase (G-6-Pase) activity. However, mice appeared to be entirely insensitive to an identical dose of APAP given either at 08:00 or 14:00 h. Hepatic glutathione (GSH) level was significantly higher at 08:00, but no difference in GSH levels between 14:00 and 20:00 h was observed in normal mice. APAP and its metabolites in blood were monitored using HPLC for 3 h following the treatment. There were no significant differences in the plasma concentrations of APAP, APAP-glucuronide, APAP-sulfate, or APAP-mercapturate among the mice treated with this drug at 08:00, 14:00 or 20:00 h. However, the APAP-cysteine and APAP-GSH levels measured at 1 h following the APAP treatment were significantly lower in mice treated with this analgesic either at 14:00 or 20:00 h. In vitro hepatic microsomal p-nitrophenol hydroxylase activities were not different between 08:00, 14:00 and 20:00 h. But ethoxyresorufin O-deethylase and aminopyrine N-demethylase activities measured at 14:00 h were significantly lower than those of 08:00 or 20:00 h. Thus, the greater hepatotoxicity of APAP administered at 20:00 h appears to be related to the marked decrease in hepatic GSH at this time period, whereas the simultaneous reduction in APAP activation may be responsible for the lack of hepatotoxicity in mice treated with this analgesic at 14:00 h. These results suggest that the temporal variation in hepatotoxicity and metabolism of APAP is determined by interactions of multiple factors including the hepatic GSH level and drug metabolizing activities.