PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC2 is an unusual coactivator in that it is the smallest coactivator identified so far, with a molecular weight of 16 kDa, and interacts with nuclear receptors using a proline-rich sequence. In yeast two-hybrid assays PNRC2 interacted with orphan receptors SF1 and estrogen receptor-related receptor alpha1 in a ligand-independent manner. PNRC2 was also found to interact with the ligand-binding domains of estrogen receptor, glucocorticoid receptor, progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X receptor in a ligand-dependent manner. A functional activation function 2 domain is required for nuclear receptors to interact with PNRC2. Using the yeast two-hybrid assay, the region amino acids 85-139 was found to be responsible for the interaction with nuclear receptors. This region contains an SH3 domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL). A mutagenesis study has shown that the SH3 domain-binding motif is important for PNRC2 to interact with all the nuclear receptors tested. Our results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator. These two proteins represent a new type of nuclear receptor co-regulatory proteins.