Sudoxicam and meloxicam are nonsteroidal anti-inflammatory drugs (NSAIDs) from the enol-carboxamide class. While the only structural difference between the two NSAIDs is the presence of a methyl group on the C5-position of the 2-carboxamidothiazole motif in meloxicam, a marked difference in their toxicological profile in humans has been discerned. In clinical trials, sudoxicam was associated with several cases of severe hepatotoxicity that led to its discontinuation, while meloxicam has been in the market for over a decade and is devoid of hepatotoxicity. In an attempt to understand the biochemical basis for the differences in safety profile, an in vitro investigation of the metabolic pathways and covalent binding of the two NSAIDs was conducted in NADPH-supplemented human liver microsomes. Both compounds demonstrated NADPH-dependent covalent binding to human liver microsomes; however, the extent of binding of [(14)C]-meloxicam was approximately 2-fold greater than that of [(14)C]-sudoxicam. While inclusion of glutathione (GSH) in microsomal incubations resulted in a decrease in covalent binding for both NSAIDs, the reduction in binding was more pronounced for meloxicam. Metabolite identification studies on [(14)C]-sudoxicam in NADPH-supplemented human liver microsomes indicated that the primary route of metabolism involved a P450-mediated thiazole ring scission to the corresponding acylthiourea metabolite (S3), a well-established pro-toxin. The mechanism of formation of S3 presumably proceeds via (a) epoxidation of the C4-C5-thiazole ring double bond, (b) epoxide hydrolysis to the corresponding thiazole-4,5-dihydrodiol derivative, which was observed as a stable metabolite (S2), (c) ring opening of the thiazole-4,5-dihydrodiol to an 2-oxoethylidene thiourea intermediate, and (d) hydrolysis of the imine bond within this intermediate to yield S3. In the case of meloxicam, the corresponding acylthiourea metabolite M3 was also observed, but to a lesser extent; the main route of meloxicam metabolism involved hydroxylation of the 5'-methyl group, a finding that is consistent with the known metabolic fate of this NSAID. Inclusion of GSH led to a decrease in the formation of M3 with the concomitant formation of an unusual two-electron reduction product (metabolite M7). The formation of M7 is proposed to arise via reduction of the imine bond in 2-oxopropylidene thiourea, an intermediate in the thiazole ring scission pathway in meloxicam. In conclusion, the results of our analysis suggest that if the covalent binding of the two NSAIDs is important to the overall hepatotoxicity risk, the differences in metabolism (differential preponderance of formation of the acylthiourea relative to total metabolism), differential effects of GSH on covalent binding, and finally differences in daily doses of the two NSAIDs may serve as a plausible explanation for the marked differences in toxicity.