Although it is well documented that neutrophils are critical for the delayed phase of hepatic ischemia-reperfusion injury, direct evidence for a specific neutrophil-derived oxidant stress in vivo is still missing. Therefore, we used a model of 60 min partial hepatic ischemia and 0-24h of reperfusion to investigate neutrophil accumulation and to analyze biomarkers for a general oxidant stress (glutathione disulfide, GSSG; malondialdehyde, MDA) and for a neutrophil-specific oxidant stress (hypochlorite (HOCl)-modified epitopes) in rats. Plasma alanine transaminase activities and histology showed progressively increasing liver injury during reperfusion. Hepatic GSSG and soluble MDA levels were elevated at 1 h of reperfusion. At that time, few neutrophils were present in sinusoids. However, the number of hepatocytes positively stained for HOCl-modified epitopes increased from 6-24h of reperfusion, which correlated with the bulk of hepatic neutrophil accumulation and extravasation into the parenchyma. Consistent with a higher oxidant stress at later time points, hepatic GSSG and protein-bound MDA levels further increased. Treatment with the NADPH oxidase inhibitor diphenyleneiodonium chloride attenuated the postischemic oxidant stress (GSSG, protein-bound MDA, hepatocytes positively stained for HOCl-modified epitopes) and liver injury at 24h of reperfusion. Ischemic preconditioning suppressed all oxidant stress biomarkers, liver injury and also extravasation of neutrophils. In conclusion, extravasated neutrophils generate HOCl, which diffuses into hepatocytes and causes oxidative modifications of intracellular proteins during the neutrophil-mediated reperfusion injury phase. Ischemic preconditioning is an effective intervention to reduce the overall inflammatory response and, in particular, to limit the cytotoxic activity of neutrophils during the later reperfusion period.