Using the DNA-binding domain (DBD) and the hinge region of human PPAR as bait in yeast two-hybrid screen, we isolated partial cDNA identical to that of the C-terminal of KIAA1769. KIAA1769 encodes a 2,080-amino acid protein (MW: 231 kDa) that was recently identified to interact with PPAR and termed PPAR-interacting cofactor 285 (here referred to as PPAR-DBD-interacting protein1, PDIP1). PDIP1 mRNA was expressed in 3T3-L1 adipocytes and THP-1 macrophages. We also identified the expression of the N terminal extended form of PDIP1 (referred to as PDIP1) consisting of 2,649 amino acids (295 kDa) in human cultured cell lines by RT-PCR and 5' RACE. RNase protection assay revealed that PDIP1 mRNA was expressed more abundantly than PDIP1 mRNA. The C-terminal region of PDIP1 directly binds DBD of PPAR, and multiple LXXLL motifs in PDIP1 were not required for the interaction. PDIP1 and similarly enhanced PPAR-mediated transactivation in transfection assays and short interfering RNA targeting PDIP1 mRNA significantly reduced transactivation by PPAR. No potent intrinsic activation domain was identified in either PDIP1 isoforms in mammalian one-hybrid assays and mutation of all LXXLL motifs did not affect enhancement of PPAR-mediated transactivation. PDIP1 and similarly augmented transactivation by PPAR, PPAR, TR1, TR1, and RXR. PDIP1 also enhanced ER- and AR-mediated transactivation, whereas PDIP1 did not. PDIP1 showed receptor-specific synergism with activation function (AF) 2-interacting coactivators in PPAR- and TR1-mediated transactivation. Together, PDIP1 might function as a transcriptional cofactor for a broad range of nuclear receptors, possibly in collaboration with specific AF2 interacting coactivators.