PPARalpha and HNF4alpha are nuclear receptors that control gene transcription by direct binding to specific nucleotide sequences. Using transgenic mice deficient for either PPARalpha or HNF4alpha, we show that the expression of the peroxisomal 3-keto-acyl-CoA thiolase B (Thb) is under the dependence of these two transcription factors. Transactivation and gel shift experiments identified a novel PPAR response element within intron 3 of the Thb gene, by which PPARalpha but not HNF4alpha transactivates. Intriguingly, we found that HNF4alpha enhanced PPARalpha/RXRalpha transactivation from TB PPRE3 in a DNA-binding independent manner. Coimmunoprecipitation assays supported the hypothesis that HNF4alpha was physically interacting with RXRalpha. RT-PCR performed with RNA from liver-specific HNF4alpha-null mice confirmed the involvement of HNF4alpha in the PPARalpha-regulated induction of Thb by Wy14,643. Overall, we conclude that HNF4alpha enhances the PPARalpha-mediated activation of Thb gene expression in part through interaction with the obligate PPARalpha partner, RXRalpha.