Most karyophilic proteins are transported into the nucleus through the importin-mediated pathway. Importin alpha acts as a receptor for classical nuclear localization signal (NLS)-containing proteins. At present, the existence of several isoforms of importin alpha in mammals is known. In this study we report on the generation of a rat monoclonal antibody (MAb) 2D9 to importin alpha NPI-1 subfamily members (importin alpha5/NPI-1 and importin alpha7/S2) using the rat lymph node method and the characterization of this antibody. In several different cultured cell extracts, MAb 2D9 reacted to endogenous NPI-1 subfamily in Western blotting experiments. Epitope mapping using recombinant deletion mutants indicated that MAb 2D9 recognized arm motif in importin alpha5/NPI-1. Using immunofluorescence, MAb 2D9 detected NPI-1 subfamily in the cytoplasm of HeLa cells. Moreover, endogenous NPI-1 subfamily was dominantly localized in the nuclei of H(2)O(2)-treated HeLa cells, suggesting that NPI-1 subfamily accumulates in the nucleus in response to oxidative stress, like importin alpha1/Rch1.