Importin proteins regulate access to the nucleus by recognizing and transporting distinct cargo proteins. Building on studies in Drosophila and Caenorhabditis elegans, we hypothesized that regulated expression and subcellular localization of specific importins may be linked to mammalian gonadal differentiation. We identified distinct developmental and cellular localization patterns for importins beta1, alpha3, alpha4 and RanBP5 (importin beta3) in fetal and postnatal murine testes using Western blotting and immunohistochemistry. Importin beta1 protein is detected in selected germ and somatic cells in fetal gonads, with a striking perinuclear staining evident from embryonic day (E) 14.5 within testicular gonocytes. RanBP5 exhibits age- and gender-specific subcellular localization within fetal gonads. At E12.5, RanBP5 protein is cytoplasmic in gonocytes but predominantly nuclear in oogonia, but by E14.5 RanBP5 appears nuclear in gonocytes and cytoplasmic in oogonia. In postnatal testes, importin alpha3 and alpha4 in spermatocytes, spermatids, and Sertoli cells display cytoplasmic and nuclear localization, respectively.