Importin alpha proteins function as adaptors to connect a cargo protein and importin beta1 in the classical nuclear import pathway. Here we measure for the first time the stoichiometry of importins alpha2, alpha3, alpha4, and beta1 in primary cells corresponding to 2 successive stages of rat spermatogenesis: meiotic spermatocytes and haploid round spermatids. Importin alpha2 levels were more than 2-fold higher in spermatocytes than in spermatids, while importins alpha4 and beta1 levels did not differ significantly. We performed a comprehensive proteomics analysis to identify binding proteins in spermatocytes and spermatids using recombinant importin alpha2 and alpha4 proteins. Among the 100 candidate partners, 42 contained a strong classical nuclear localization signal (cNLS; score of>6 by cNLS Mapper), while 8 nuclear proteins lacked any cNLS. In addition, we developed a new strategy to predict which cargoes bind to importin alpha through the conserved C-terminal acidic domain (ARM repeats 9-10), and provided functional validation of a predicted importin alpha C-terminal binding segment in Senataxin and Smarca4. Evaluation of this set of candidate binding partners from spermatogenic cells using several bioinformatics approaches provides new evidence that individual importin alphas may serve unique and nonredundant roles in mediating cellular differentiation.-Arjomand, A., Baker, M. A., Li, C., Buckle, A. M., Jans, D. A., Loveland, K. L., Miyamoto, Y. The alpha-importome of mammalian germ cell maturation provides novel insights for importin biology.