In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Ga14-PPARγ as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator- 1 (SRC-1) as a PPAR transcriptional coactivator. We now report the isolation of a cDNA encoding a 165-kDa PPARγ-binding protein, designated PBP which also serves as a coactivator. PBP also binds to PPARα, RARα, RXR, and TRβ1, and this binding is increased in the presence of specific ligands. Deletion of the last 12 amino acids from the carboxyl terminus of PPARγ results in the abolition of interaction between PBP and PPARγ. PBP modestly increased the transcriptional activity of PPARγ, and a truncated form of PBP (amino acids 487-735) acted as a dominant-negative repressor, suggesting that PBP is a genuine coactivator for PPAR. In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. In situ hybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation.