Peroxisome proliferator-activated receptor (PPAR) forms a heterodimer with retinoid X receptor (RXR) that binds to the peroxisome proliferator responsive element (PPRE) to regulate the expression of target genes. PPRE of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) gene has previously been shown to consist of three imperfect TGACCT half-sites and form two distinct complexes (C1 and C2) with the nuclear extracts from H4IIEC3 cells. The present study identifies another imperfect TGACCT motif involved in the PPAR/RXR-mediated trans-activation process and demonstrates that these four imperfect TGACCT motifs constitute an unique binding site consisting of two DR1 elements overlapping a DR2 element. PPAR and RXR cooperatively bind the two DR1 elements to form C1 complex or bind DR2 element to form C2 complex with a 1:1 ratio. Saturation of the HD PPRE probes with receptor proteins cannot convert the heterodimeric C2 complex to the higher order C1 complex, suggesting that they are formed independently. Transfection analyses indicate that mutation of any one of these TGACCT motifs or truncation of the entire HD PPRE into a separate DR1 and DR2 element significantly reduced the transcriptional response of HD PPRE to peroxisome proliferators. The rat HD PPRE differentially binds with one or two PPAR/RXR heterodimers providing the peroxisome proliferator signaling pathway with two levels of response.