Direct repeat motifs composed of two hexamer half-sites spaced by a single nucleotide (DR-1) are recognized by several members of the nuclear hormone receptor superfamily. We examined, by means of gene transfection assays, the interplay between the DR-1-binding nuclear receptors commonly expressed in liver, peroxisome proliferator-activated receptor alpha (PPARalpha), hepatocyte nuclear factor-4 (HNF-4), and chicken ovalbumin upstream transcription factor I (COUP-TFI). Both PPARalpha and HNF-4 efficiently bound to the acyl-CoA oxidase gene enhancer element, but PPARalpha exhibited much stronger transactivation than HNF-4. As a result, HNF-4 suppressed the gene-activating function of PPARalpha, when they were expressed together, due to competition for a common binding site. On the other hand, HNF-4, but not PPARalpha, effectively bound to the apolipoprotein CIII gene element, and activated gene transcription. PPARalpha had no effect even when co-expressed with HNF-4. COUP-TFI bound to both elements, and suppressed the gene activation by PPARalpha and HNF-4. Thus, these nuclear receptors have individual functions in gene regulation, and exhibit complex compound effects when they co-exist.