The mass spectra of several thioether conjugates of the widely used analgesic, acetaminophen (4'-hydroxyacetanilide), have been recorded under various ionization conditions. Conjugates were obtained from both in vitro and in vivo sources and purified by high performance liquid chromatography. Some standards were chemically synthesized. Of the thioethers examined, only the methylthio and mercapturic acid conjugates provided parent ions under electron impact conditions. In the chemical ionization mode, using isobutane as the reagent gas, the cysteinyl conjugate gave a pseudomolecular ion as well, although relatively large quantities (10-20 micrograms) of this amino acid adduct were required. Because of the highly polar nature and thermal instability of the cysteinyl and glutathionyl conjugates, these two thioethers were most successfully analyzed by field desorption techniques. Field desorption mass spectrometry was well suited for direct analysis of these two adducts where prominent [M]+, [MH]+ or [M + Na]+ ions were observed. Furthermore, by application of the field desorption/collision induced dissociation and linked (B/E) scan technique, structurally informative fragmentation patterns were generated. In addition, field desorption mass spectrometry was used successfully to characterize the glucuronide conjugate of acetaminophen but not the sulfate conjugate.