Sulfation of acetaminophen is a high-affinity and low-capacity conjugation pathway in rats. It is thought that sulfation becomes saturated in rats at high doses of acetaminophen because of limited availability of the active sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the supply of which is, in turn, limited by the availability of its precursor, inorganic sulfate. The present study was designed to determine whether a similar mechanism is responsible for capacity-limited sulfation in mice. Saturation of acetaminophen sulfation occurs in both species; however, at the maximal rate of sulfation, sulfate and PAPS concentrations were markedly decreased in rats but not in mice. Administration of sodium sulfate and the sulfate precursor N-acetylcysteine enhanced the formation of acetaminophen sulfate in rats, but not in mice. Mice exhibited lower activities of hepatic PAPS synthetic enzymes (i.e., ATP sulfurylase and APS kinase) and sulfotransferase than rats, which may in part be responsible for their lower capacity to sulfate acetaminophen. In addition, administration of acetaminophen further decreased phenolsulfotransferase activity in mice. In rats, administration of acetaminophen did not influence hepatic sulfotransferase activity. These observations suggest that while the capacity of rats to sulfate acetaminophen is limited by the availability of PAPS, in mice it is limited by sulfotransferase activity.