Certain functional groups/structural motifs are known to generate chemically reactive metabolites that can covalently modify essential cellular macromolecules and, therefore, have the potential to disrupt biological function and elicit idiosyncratic adverse drug reactions. In this report, we describe the bioactivation of 5-substituted 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles, which can be added to the growing list of structural alerts. When 5-substituted 2-(methylthio)-1,3,4-thiadiazoles and 2-(methylthio)-1,3-benzothiazole were incubated with pooled human liver microsomes in the presence of NADPH and GSH, unusual GSH adducts were formed. Characterization of these GSH adducts by high-resolution mass spectrometry indicated the replacement of the methylthio- group by GSH, and NMR experiments ascertained the proposed structures. On the basis of the metabolic profile change in incubation samples with/without GSH, we proposed that the GSH adduct formation involved two steps: (1) enzymatic oxidation of the alkylthio- group to form sulfoxide and sulfone and (2) nucleophilic displacement of the formed sulfoxide and sulfone by GSH. The proposed mechanism was confirmed by the formation of the same GSH adduct from the incubation of synthetically prepared sulfoxide and sulfone compounds in buffer. We found the sulfur oxidation step was significantly inhibited (80-100%) by preincubation with 1-aminobenzotriazole but was much less affected by thermoinactivation (0-45%), suggesting that the sulfoxidation step is primarily catalyzed by cytochrome P450s and not by flavin monooxygenases. We also investigated the presence of this bioactivation pathway in more than a dozen compounds containing 2-(alkylthio)-1,3,4-thiadiazole and 2-(alkylthio)-1,3-benzothiazoles. The common GSH adduct formation pathway demonstrated by current studies raises a new structural alert and potential liability in drug safety when 2-alkylthio derivatives of 1,3-benzothiazoles and 1,3,4-thiadiazoles are incorporated in drug design.