Kupffer cells and polymorphonuclear leukocytes (PMNs) contribute to the severe reperfusion injury of the liver after ischemia at different time points. The objective of this study was to identify die cellular source(s) of reactive oxygen formation during the PMN‐ induced injury phase. Kupffer cells and PMNs were isolated from the liver after 45 min of ischemia and 5 h or 24 h of reperfusion using collagenase‐pronase digestion and a centrifugal elutriation method. Spontaneous superoxide anion (O₂⁻) formation by large Kupffer cells (basal value 0.65 ± 0.16 nmol/h/10⁶ cells) was increased (up to 550%) during the entire reperfusion period. No enhanced O₂⁻ generation by the small Kupffer cell fraction was observed at any time. Control PMNs generated only small amounts of O₂⁻ spontaneously (0.25 ± 0.05 nmol O₂⁻/h/10⁶ cells), but hepatic PMNs generated significantly more superoxide: 1.90 ± 0.58 nmol O₂⁻/h/10⁶ cells at 5 h and similarly at 24 h of reperfusion. All cell types were significantly primed for enhanced O₂⁻ formation during reperfusion; the priming effect was consistantly higher for stimulation with opsonized zymosan (receptor‐ mediated signal transduction pathway) compared to phor‐ bol myristate acetate (protein kinase C activation). Our data support the hypothesis that PMNs and large Kupffer cells are predominantly responsible for the postischemic oxidant stress during the later reperfusion injury phase after hepatic ischemia in viva.