We have identified a peroxisome proliferator response element (PPRE) approx. 4300 nucleotide upstream of the rat cytochrome P-450 CYP4A1 gene. Two members of the steroid-hormone-receptor superfamily, the peroxisome proliferator-activated receptor-alpha (PPAR alpha) and the retinoid X receptor-alpha (RXR alpha), bind specifically to this element as a heterodimer, and this element confers responsiveness to the peroxisome proliferator Wyeth-14,643 when tested in co-transfection assays. A second element, located 35 nucleotides further upstream, fails to bind PPAR alpha/RXR alpha heterodimers and is unresponsive to Wy-14,643 in co-transfection assays. Both elements are, however, responsive to 9-cis-retinoic acid in the presence of RXR alpha, when tested in the co-transfection assay. As RXR alpha fails to bind to either element as a homodimer, we suggest that RXR alpha interacts with PPAR alpha to regulate transcription via the proximal element, and interacts with some other cellular factor to regulate transcription via the more distal element. This is consistent with previous reports that a number of peroxisome proliferator-regulated genes contain PPRE-like elements as part of their regulatory sequences, which may be recognized by several receptor combinations. This provides further evidence that PPARs and their co-factors are important in mediating the pleiotropic action of peroxisome proliferators.