Purpose <p>Cytochrome P450 (P450) metabolites of arachidonic acid (AA) are potential therapeutic targets in ischemia–reperfusion (IR) injury, yet their disposition and actions in hepatic IR are unknown. Cellular AA and its P450 metabolites are primarily bound to lipid membranes, resulting in negligible free concentrations. This study investigates how hepatic IR injury impacts the disposition of free AA and its major P450 metabolites in rats.</p> Methods <p>Rats underwent 60 min of partial (70%) ischemia or sham surgery, followed by 5 min, 3 h, or 24 h of reperfusion. The effects of IR injury on free concentrations of AA and its major P450-generated metabolites in the liver were studied using LC–MS/MS. The activities of phospholipase A₂ (PLA₂) enzymes, which release AA and its preformed metabolites from lipid membranes, were also measured in the liver and plasma. Additionally, <i>in vitro</i> rates of metabolite formation and the levels of major AA-metabolizing enzymes were assessed in microsomes.</p> Results <p>Hepatic IR injury significantly increased free liver concentrations of AA (sevenfold) and its metabolites (1.8–9.1-fold) only in the 5-min group. Similarly, PLA₂ enzyme activity in the liver and plasma was higher only in this group. Conversely, liver IR injury caused a significant reduction or no change in the <i>in vitro</i> rate of AA metabolism to P450-mediated metabolites in the 5-min group.</p> Conclusions <p>Liver IR injury rapidly increases liver concentrations of free P450-mediated metabolites of AA, likely due to IR-induced release of preformed metabolites and AA from lipid membranes, rather than an increase in P450 enzymatic activity.</p>