The objective of this study was to assess the role of inducible nitric oxide synthase (iNOS) in ischemia- and reperfusion (I/R)-induced liver injury. We found that partial hepatic ischemia involving 70% of the liver resulted in a time-dependent increase in serum alanine aminotransferase (ALT) levels at 1–6 h following reperfusion. Liver injury at 1, 3, and 6 h post-ischemia was not due to the infiltration of neutrophils as assessed by tissue myeloperoxidase (MPO) activity and histopathology. iNOS-deficient mice subjected to the same duration of ischemia and reperfusion showed dramatic and significant increases in liver injury at 3 but not 6 h following reperfusion compared to their wild type controls. Paradoxically, iNOS mRNA expression was not detected in the livers of wild type mice at any point during the reperfusion period and pharmacological inhibition of iNOS using l -N⁶(iminoethyl)-lysine (l -NIL) did not exacerbate post-ischemic liver injury at any time post-reperfusion. These data suggest that iNOS deficiency produces unanticipated genetic alterations that renders these mice more sensitive to liver I/R-induced injury.