Sulfotransferase (ST) is considered to be generally not induced by xenobiotics. However, it has been reported that steroids such as dexamethasone (DEX) and pregnenolone-16a-carbonitrile (PCN) are effective ST inducers in rats, and sulfation of xenobiotics is quite different in rats and mice. The present study is primarily aimed at determining the effect of sulfate and steroids on the metabolism of acetaminophen (AA) in vitro using monolayer cultured hepatocytes of Sprague-Dawley rats and ICR mice. Hepatocytes of rats and mice were incubated with inorganic sulfate (0.25, 0.5, 1.0, 2.0, 4.0 mM) and AA in SO4-depleted media. AA sulfation rates increased as the sulfate concentration was raised to 1.0 mM in rats, whereas the addition of inorganic sulfate to the media had a lesser effect in mice hepatocytes. After pretreatment with DEX (0.1, 1.0, 10, 100 microM) and PCN (0.1, 1.0, 10 microM) for 3 d, hepatocytes were incubated with AA in media containing 4.0 mM SO4-. Pretreatment of the hepatocytes with DEX caused an increase in the glucuronidation and sulfation of AA by 2-3 folds in rats, but to a lesser extent in mice. PCN significantly enhanced the formation of AA-glucuronide and AA-sulfate in mice, but had a minimal effect on rat hepatocytes. In summary, sulfate and DEX markedly enhanced the formation of AA-sulfate in rats hepatocytes, and DEX and PCN increased the sulfation of AA in mice hepatocytes. These results partially support the claim that DEX and PCN are effective ST and uridine diphosphate-glucuronyltransferase inducers in vivo.