Peroxisomal proliferators, such as ciprofibrate, are used extensively as effective hypolipidemic drugs. The effects of these compounds on lipid metabolism require ligand binding activation of the peroxisome proliferator-activated receptor (PPAR) alpha subtype of nuclear receptors and involve transcriptional activation of the metabolic pathways involved in lipid oxidative metabolism, transport, and disposition. omega-Hydroxylated-eicosatrienoic acids (HEETs), products of the sequential metabolism of arachidonic acid (AA) by the cytochrome P450 CYP2C epoxygenase and CYP4A omega-hydroxylase gene subfamilies, have been identified as potent and high-affinity ligands of PPARalpha in vitro and as PPARalpha activators in transient transfection assays. Using isolated rat hepatocytes in culture, we demonstrate that specific inhibition of either the CYP2C epoxygenase or the CYP4A omega-hydroxylase abrogates ciprofibrate-induced peroxisomal proliferation, whereas inhibition of other eicosanoid-synthesizing pathways had no effect. Conversely, overexpression of the rat liver CYP2C11 epoxygenase leads to spontaneous peroxisomal proliferation, an effect that is reversed by a CYP inhibitor. Based on these results, we propose that HEETs may serve as endogenous PPARalpha ligands and that the P450 AA monooxygenases participate in ciprofibrate-induced peroxisomal proliferation and the activation of PPARalpha downstream targets.