The family of insulin receptor substrates (IRSs) has been reported to play important roles for signal transduction of various hormones. Four members of the IRS family have been described. Each IRS is believed to have different functions; however, the distinct physiological roles of each IRS are unclear. This study was undertaken to determine the intracellular localization of IRS-3. IRS-3 was expressed in COS-7 cells as fusion with a green fluorescent protein (GFP), and subcellular localization of the chimera protein was analyzed by fluorescent microscopy. Surprisingly, GFP-IRS-3 was localized not only adjacent to the plasma membrane but also in the nucleus. We confirmed by immunostaining with anti-IRS-3 antibody that non-fused IRS-3 protein is also localized in the nucleus of COS-7 cells that were transfected with IRS-3 cDNA. In addition, we detected endogenous IRS-3 in the nucleus of isolated rat adipocytes. We then studied subcellular localization of deletion mutants and fragments of IRS-3 fused with GFP. We found that the region corresponding to amino acid residues 192-223 in the phosphotyrosine binding domain played an important role in nuclear localization. This region includes sequences that are unique to IRS-3. We then investigated intracellular localization of other IRSs fused with GFP. GFP-IRS-1, GFP-IRS-2, and GFP-IRS-4 were mainly localized in the cytosol or plasma membranes. Chimeric protein, Gal4 DNA binding domain fused with IRS-3 C-terminal region, increased transcription of the reporter gene containing Gal4 binding site in human embryonic kidney 293 cells. These results suggest that intracellular localization of IRS-3 is determined by a different mechanism from other IRS proteins, and that IRS-3 possesses a transcription-regulating activity.