Flutamide (FLU) is a competitive antagonist of the androgen receptor which has been reported to induce severe liver injury in some patients. Several experimental models suggested that an episode of inflammation during drug treatment predisposes animals to tissue injury. The molecular cytotoxic mechanisms of FLU in isolated rat hepatocytes using an in vitro oxidative stress inflammation system were investigated in this study. When a nontoxic hydrogen peroxide (H2O2) generating system (glucose/glucose oxidase) with peroxidase or iron(II) [Fe(II)] (to partly simulate in vivo inflammation) was added to the hepatocytes prior to the addition of FLU, increases in FLU-induced cytotoxicity and lipid peroxidation (LPO) were observed that were decreased by 6-N-propyl-2-thiouracil or deferoxamine, respectively. N-Acetylcysteine decreased FLU-induced cytotoxicity in this system. Potent antioxidants, for example, Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), resveratrol (3,5,4′-trihydroxy-trans-stilbene), and DPPD (N,N′-diphenyl-1,4-phenylenediamine) also significantly decreased FLU-induced cytotoxicity and LPO and increased mitochondrial membrane potential (MMP) and glutathione (GSH) levels in the H2O2 generating system with peroxidase. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known reactive oxygen species (ROS) scavenger and superoxide dismutase mimetic, also significantly decreased toxicity caused by FLU in this system. These results raise the possibility that the presence or absence of inflammation may be another susceptibility factor for drug-induced hepatotoxicity.