Ischemia/reperfusion (I/R) is an important problem in liver resection and transplantation that is associated with hepatocellular dysfunction and injury. This study was designed to investigate whether a difference in hepatocyte susceptibility occurs in the periportal (PP) and/or perivenous (PV) zones in response to hypoxia/reoxygenation (H/R), and to delineate the mechanisms underlying this susceptibility. H/R was induced in an in situ perfused mouse liver model with deoxygenated Krebs‐Henseleit buffer followed by oxygenated buffer. Selective destruction of PP or PV sites was achieved by digitonin perfusion into the portal or inferior vena cava, and was confirmed by histological evaluations and zone‐specific enzymes. Hepatocellular injury was assessed by alanine aminotransferase (ALT) release. In whole liver, H/R significantly increased perfusate ALT. H/R of PP‐enriched zones caused ALT release that was similar to that of whole liver (80 + 10 vs. 70 + 12 U/mg protein), consistent with significant PP hepatocyte injury. Minimal ALT release occurred in PV zones (10 + 5 U/mg protein). Administration of N‐acetyl L‐cysteine or a chimeric superoxide dismutase (SOD)—SOD2/3, a genetically engineered SOD—abrogated ALT release in H/R‐perfused PP zones, implicating a role for superoxide (O). This elevated ALT release was attenuated by gadolinium chloride pretreatment, indicating that Kupffer cells are the O source. Enzymatic inhibition of cellular nitric oxide synthase (NOS) or genetic depletion of endothelial nitric oxide synthase (eNOS) aggravated hypoxia injury while exogenous NO and inducible nitric oxide synthase (iNOS) deficiency abolished reoxygenation injury. In conclusion, PP hepatocytes are more vulnerable to H/R; this injury is mediated directly or indirectly by Kupffer cell derived O and is limited by eNOS‐derived NO. (HEPATOLOGY 2004;39:1544–1552.)