Our previous studies demonstrated marked sex differences in the metabolism of acetaminophen in rats both in vivo and in hepatocyte culture. This study examined the sex differences in human acetaminophen metabolism, and the ability to reproduce in vivo metabolism in human hepatocyte cultured on Matrigel vs. type 1 collagen. Human hepatocytes were isolated by collagenase perfusion of 10-15 g biopsies of patients without liver disease undergoing elective abdominal operations (8 females, age 41.3 +/- 19.3 years; 6 males, age 47.7 +/- 21.3 years). Postoperatively, patients were given 1 g of acetaminophen orally and a 24-hr urine collected to determine the metabolic fate. There were no sex differences in acetaminophen conjugation in vivo, nor in the hepatic acetaminophen sulfotransferase activities as observed in the rodents. Hepatocytes were cultured with acetaminophen (0, 150, 250, 500, and 1000 microM) on Matrigel and type I collagen. Acetaminophen glucuronidation predominated over acetaminophen sulfation just as in vivo without sex differences on days 1 and 2 in culture. By days 3 and 4, however, glucuronidation by female hepatocytes became enhanced compared with males. With increasing acetaminophen concentration (dose), there was a linear increase in sulfate and glucuronide conjugation without saturation of either pathway as observed in the rat. CONCLUSIONS 1) there are no sex differences in acetaminophen metabolism or acetaminophen sulfotransferase activity as observed in the rat; 2) acetaminophen sulfation and glucuronidation by cultured human hepatocytes in vitro replicated in vivo metabolism on matrigel and type 1 collagen for the first 2 days in culture; and 3) glucuronidation became enhanced with time in the culture of female, but not male hepatocytes.