Two catalytic isoforms of the Na,K-ATPase, α1 and α4, are present in testis. While α1 is ubiquitously expressed in tissues, α4 predominates in male germ cells. Each isoform has distinct enzymatic properties and appears to play specific roles. To gain insight into the relevance of the Na,K-ATPase α isoforms in male germ cell biology, we have studied the expression and activity of α1 and α4 during spermatogenesis and epididymal maturation. This was explored in rat testes at different ages, in isolated spermatogenic cells and in spermatozoa from the caput and caudal regions of the epididymis. Our results show that α1 and α4 undergo differential regulation during development. Whereas α1 exhibits only modest changes, α4 increases with gamete differentiation. The most drastic changes for α4 take place in spermatocytes at the mRNA level, and with the transition of round spermatids into spermatozoa for expression and activity of the protein. No further changes are detected during transit of spermatozoa through the epididymis. In addition, the cellular distribution of α4 is modified with development, being diffusely expressed at the plasma membrane and intracellular compartments of immature cells, finally to localize to the midregion of the spermatozoon flagellum. In contrast, the α1 isoform is evenly present along the plasma membrane of the developing and mature gametes. In conclusion, the Na,K-ATPase α1 and α4 isoforms are functional in diploid, meiotic and haploid male germ cells, α4 being significantly upregulated during spermatogenesis. These results support the importance of α4 in male gamete differentiation and function.