Exposure to a nontoxic dose of bacterial endotoxin (lipopolysaccharide [LPS]) potentiates the hepatotoxicity of aflatoxin B1 (AFB1). Because some of the pathophysiologic effects associated with LPS are mediated through tumor necrosis factor α (TNF-α), this study was conducted to explore the role of TNF-α in the AFB1/LPS model. Male Sprague-Dawley rats (250-300 g) were treated with either 1 mg AFB1/kg, intraperitoneally, or its vehicle (0.5% dimethyl sulfoxide [DMSO]/water), and 4 hours later with either Escherichia coli lipopolysaccharide (7.4 × 106EU/kg, intravenously) or its saline vehicle. LPS administration resulted in a marked rise in TNF-α levels at 6 hours, which preceded the onset of liver injury. TNF-α messenger RNA (mRNA) in liver was increased by LPS treatment. The mRNA of receptors (R1 and R2) for TNF-α was also examined. R1 mRNA levels were not altered; however, R2 mRNA levels were increased by either AFB1 or LPS administration. To determine if TNF-α plays a causal role in the development of liver injury, the increase in TNF-α was attenuated by administration of either pentoxifylline or anti-TNF-α serum, and liver injury was assessed. Administration of either of these agents resulted in protection. LPS treatment resulted in the upregulation of gene transcription for cyclooxygenase-2 (COX-2). However, administration of the selective COX-2 inhibitor NS-398 did not decrease injury. TNF-α and COX-2 inhibitors did not affect hepatic sequestration of neutrophils. Furthermore, it did not appear that TNF-α contributed to injury through inhibition of tissue repair. These data support the hypothesis that LPS-induced expression of TNF-α underlies the potentiation of AFB1-induced hepatotoxicity.