Peroxisome proliferator-activated receptors (PPARs) stimulate the expression of several genes involved in lipid metabolism by binding to specific cis-acting peroxisome proliferator-responsive elements (PPREs) via cooper-ativity with retinoid X receptors. We demonstrate here that hepatocyte nuclear factor-4 (HNF-4), another member of the nuclear hormone receptor superfamily, bound with differing affinities to the PPREs from the genes encoding rat acyl-CoA oxidase and hydratase-dehydrogenase, the first two enzymes of the peroxisomal beta-oxidation pathway. In cotransfection assays, HNF-4 repressed rat PPAR-dependent activation of a reporter gene linked to the acyl-CoA oxidase PPRE, either in the absence or presence of the peroxisome proliferator, Wy-14,643. Rat PPAR-dependent activation of a reporter gene linked to the hydratase-dehydrogenase PPRE was less efficiently repressed by HNF-4 in the absence of Wy-14,643 than was activation from the acyl-CoA oxidase PPRE. However, in the presence of Wy-14,643, HNF-4 functioned cooperatively with PPAR to significantly enhanced induction from the hydratase-dehydrogenase PPRE. These results suggest that the genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway are subject to differential regulation by the interplay of multiple members of the steroid/nuclear hormone receptor superfamily, mitigated in part by the structures of the PPREs and by the presence of activators of PPARs.