This present study was designed to assess the role of metabolic and pharmacokinetic factors in the lower susceptibility of female rats compared to male rats to xenobiotics metabolized by the cytochrome P-450-dependent mixed-function oxidase (MFO) system. Adult intact male and female Sprague-Dawley rats were administered labeled acetaminophen (1 g/kg body weight + 5 microCi [3H]acetaminophen) after an overnight fast. They were bled and killed at 0.5, 1, 2, 3, 6, 12, 24, and 36 h after drug administration. The percentage of [3H]acetaminophen radioactivity remaining in blood, liver, GI tract, and excreted in the urine was determined at all time intervals. Plasma prothrombin time and serum transaminases were determined as indices of hepatotoxicity. Hepatic GSH and glycogen were assayed. Total urinary acetaminophen and its metabolites and the molar percent of various metabolites excreted during the first 6 h were determined. Castrated male and ovariectomized female rats and their respective controls were also given acetaminophen (APAP) and were killed 24 h later to determine hepatotoxicity. The extent of hepatic damage in the intact male rats was greater and appeared sooner than in the female rats. Hepatic GSH and glycogen were depleted earlier in female rats. The percent of the administered dose excreted in the urine during the first 6 h was 17.5 for the male rat versus 24.5 for the female rat. While the APAP glucuronide conjugate concentration was significantly higher, the APAP sulfate conjugate concentration was lower in the female than it was in the male rat. Although peak radioactivity in serum was reached by 30 min in both male and female rats, suggesting quick intestinal absorption, it was significantly higher in female rats and was associated with decreased intestinal and hepatic levels and increased urinary excretion when compared to male rats. While castration of male rats decreased susceptibility to hepatotoxicity, ovariectomy of female rats tended to increase susceptibility to hepatotoxicity in comparison to their respective controls. Our data suggest that aside from the reported sex differences in the cytochrome P-450-dependent MFO enzymes, there are significant differences in GSH utilization. There are also significant changes in glucuronidation and sulfation pathways, as well as in the pharmacokinetics of acetaminophen, which tend to protect female rats against acetaminophen hepatotoxicity.