Peroxisome proliferator-response elements (PPRE) are cis-acting regulatory elements that confer responsiveness to peroxisome proliferators and various fatty acids by serving as target sites for ligand-activated peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. Other cellular factors, including additional nuclear hormone receptors, also interact with PPREs and modulate PPAR function. We have developed a positive selection strategy in yeast to identify mammalian factors that functionally interact with PPREs. Saccharomyces cerevisiae containing an integrated copy of the HIS3 gene under transcriptional control of a minimal CYC1 promoter and two copies of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase PPRE was constructed and transformed with a rat liver cDNA yeast expression library. Plasmids were isolated from his + transformants. One plasmid contained a cDNA encoding the complete rat chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily. COUP-TFII potently activated PPRE-linked reporter gene expression in yeast, and COUP-TFII synthesized in yeast or in vitro formed specific protein/DNA complexes with this PPRE. Significantly, COUP-TFII did not activate transcription of PPRE-linked reporter genes in mammalian cells but rather strongly inhibited induction mediated by PPAR/RXR. Our findings demonstrate the utility of using genetic screening in yeast to identify sequence-specific DNA binding transcription factors.