In their journey from the male to the female reproductive tract, spermatozoa are confronted with a constantly changing environment. To cope with the associated challenges, spermatozoa express a distinct set of transporters, channels and pumps. One of the membrane proteins unique to spermatozoa is the alpha4 isoform of the Na+,K+-ATPase. In addition to alpha4, spermatozoa express the ubiquous alpha1 variant. To get a detailed understanding of how alpha1 and alpha4 differ, and why spermatozoa need an additional Na+,K+-ATPase, we have conducted an electrophysiological comparison of the rodent isoforms (rat alpha4 versus mouse alpha1-3) using the Xenopus oocyte expression system. We demonstrate isoform-specific differences in the voltage sensitivity of steady-state turnover, with alpha2 being the more sensitive, and alpha1 and alpha2 having faster Na+ release kinetics than alpha3 and alpha4. Our data further show that the alpha1 and alpha2 turnover rates are fast compared with those of alpha3 and alpha4. Finally, alpha4 is less influenced by changes in extracellular Na+ and temperature than alpha1. Based on these findings, we discuss the possibility that evolution has selected robust activity rather than rapid turnover for alpha4.