Dietary supplementation with the n-3 polyunsaturated fatty acids (n-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to rats preconditions the liver against ischemia-reperfusion (IR) injury, with reduction of the enhanced nuclear factor-kappa B (NF-kappa B) functionality occurring in the early phase of IR injury, and recovery of IR-induced proinflammatory cytokine response. The aim of the present study was to test the hypothesis that liver preconditioning by n-3 PUFA is exerted through peroxisone proliferator-activated receptor alpha (PPAR-alpha) activation and interference with NF-kappa B activation. For this purpose we evaluated the formation of PPAR-alpha/NF-kappa Bp65 complexes in relation to changes in PPAR-alpha activation, I kappa B-alpha phosphorylation and serum levels and expression of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha in a model of hepatic IR-injury (1 h of ischemia and 20 h of reperfusion) or sham laparotomy (controls) in male Sprague Dawley rats. Animals were previously supplemented for 7 days with encapsulated fish oil (General Nutrition Corp., Pittsburg, PA) or isovolumetric amounts of saline (controls). Normalization of IR-altered parameters of liver injury (serum transaminases and liver morphology) was achieved by dietary n-3 PUFA supplementation. EPA and DHA suppression of the early IR-induced NF-kappa B activation was paralleled by generation of PPAR-alpha/NF-kappa Bp65 complexes, in concomitance with normalization of the IR-induced IkB-alpha phosphorylation. PPAR-alpha activation by n-3 PUFA was evidenced by enhancement in the expression of the PPAR-alpha-regulated Acyl-CoA oxidase (Acox) and Carnitine-Palmitoyl-CoA transferase I (CPT-I) genes. Consistent with these findings, normalization of IR-induced expression and serum levels of NF-kappa B-controlled cytokines IL-I beta and TNF-alpha was observed at 20 h of reperfusion. Taken together, these findings point to an antagonistic effect of PPAR-alpha on NF-kappa B-controlled transcription of pro-inflammatory mediators. This effect is associated with the formation of PPAR-alpha/NF-kappa Bp65 complexes and enhanced cytosolic I kappa B-alpha stability, as major preconditioning mechanisms induced by n-3 PUFA supplementation against IR liver injury.