Bile from rats treated with acetaminophen was analyzed by direct injection onto a thermospray liquid chromatography/mass spectrometry system. Two conjugated metabolites of acetaminophen were separated by the high-pressure liquid chromatographic system and analyzed by mass spectrometry. The conjugates were identified as the glutathione-acetaminophen conjugate and the glucuronide-acetaminophen conjugate by comparison of the chromatographic retention times and the mass spectra to that of the synthetic standards. No evidence of acetaminophen metabolites was observed when bile samples were subjected to direct analysis by fast atom bombardment mass spectrometry.