A heteromeric integral membrane protein, Na /K ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na /K ATPase, to Na /K ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na /K ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na /K ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na /K ATPase. Binding of ouabain to Na /K ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na /K ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na /K ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na /K ATPase in mammalian sperm capacitation.