Previously, we demonstrated that oltipraz [OTP: 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione] prevented the hepatotoxicity of acetaminophen (AAP) in hamsters and that the observed protection was not related to increases in hepatic reduced glutathione (GSH) levels. These experiments were designed to elucidate the mechanism of OTP-induced protection with respect to an apparent non-GSH-dependent system. Marked differences in the relative amounts of hepatic GSH content depleted by AAP in control vs OTP-treated hamsters occurred. Urinary recoveries of AAP and metabolites indicated that more AAP-glucuronide was formed at the expense of other major metabolites (AAP-GSH, -N-acetylcysteine, and -sulfate) in OTP-treated hamsters, while plasma toxicokinetic modeling suggested a greater rate of AAP systemic clearance. An increased apparent formation rate constant for AAP glucuronidation (135%), in concert with significantly lower apparent formation rate constants for those metabolites which reflect the production of the reactive intermediate from AAP (glutathione and N-acetylcysteine), provide the rationale for this shift of metabolism. The biochemical basis for metabolic shunting is significantly elevated hepatic UDP-glucuronic acid content, an increased calculated UDP-glucuronic acid synthetic rate, and an increased liver microsomal UDP-glucuronyl transferase activity in OTP-treated animals. These changes in AAP conjugation were concomitant with decreased fractional clearance of AAP via bioactivation and less in vivo AAP covalent binding. These data support the hypothesis that OTP provides a protecting effect from AAP hepatotoxicity due to an augmented and predisposing glucuronidation capacity.