While the function of the ubiquitous Na,K-ATPase alpha1 subunit has been well documented, the role of the sperm-specific alpha4 isoform of this ion transporter is less known. We have explored the importance of alpha4 in rat sperm physiology by taking advantage of the high sensitivity of this isoform for the inhibitor ouabain. Using concentrations that selectively block alpha4 activity, we found ouabain to reduce not only sperm total motility, but also multiple parameters of sperm movement, including progressive motility, straight line, curvilinear, and average path velocities, lateral head displacement, beat cross frequency, and linearity. According to a direct role of alpha4 in Na(+) transport, ouabain inhibition of alpha4 increased [Na(+)](i) in the male gametes. In addition, interference of alpha4 activity with ouabain produced cell membrane depolarization, diminished pH, and increased [Ca(2)(+)](i) in spermatozoa. Inhibition of alpha4 was sufficient to cause all these effects and additional blockage of alpha1, the other Na,K-ATPase alpha isoform expressed in sperm, and higher doses of ouabain did not result in further changes in the cell parameters studied. These results show that alpha4 is the Na,K-ATPase isoform primarily involved in controlling the transmembrane Na(+) gradient in sperm, and that alpha4 activity is necessary for maintaining membrane potential, [Ca(2)(+)](i), and [H(+)](i) in the cells. The high dependence of sperm motility on membrane excitability, [Ca(2)(+)](i), and acid-base balance suggests that their regulation is the mechanism by which alpha4 maintains motility of the male gametes.