Peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is expressed predominantly in adipose tissue. Forced expression of the two isoforms of this receptor, PPARγ1 and PPARγ2, in fibroblasts initiates a transcriptional cascade that leads to the development of adipocyte phenotype. Using the yeast two-hybrid system and GAL4-PPARγ as bait to screen mouse liver cDNA library, we isolated a mouse steroid receptor coactivator (mSRC-1) involved in nuclear hormone receptor transcriptional activity as a mPPARγ interactive protein. mSRC-1 cDNA we isolated contains an open reading frame of 1447 amino acids and encodes a new member of the basic helix-loop-helix-PAS domain family. We show that the binding of mSRC-1 to mPPARγ is ligand independent and coexpression of mSRC-1 with mPPARγ increases the transcriptional activity of mPPARγ in the presence of mPPARγ ligand. We have identified the presence of two putative mPPARγ binding sites in the mSRC-1, one between residues 620 and 789, and the second between residues 1231 and 1447. These two regions exhibit different degrees of binding affinity for mPPARγ. We also show that mSRC-1 exhibits its own constitutive transcriptional activity in the yeast as well as in mammalian cells. These results suggest that mSRC-1 interacts with PPARγ and plays a role in the PPARγ-mediated signaling pathway.