The rapidly growing world population and the high rate of unintended pregnancies make contraception a need and a priority for any public health program. While several contraceptive methods for women are currently available, a more comprehensive approach to birth control requires extending contraception to males. However, a safe, effective and reversible contraceptive for men is still unavailable. An attractive approach t develop male contraceptives consists in targeting proteins that are specifically expressed in sperm and are required for sperm fertility. Evidence from our laboratory has shown that Na,K-ATPase ?4, a sperm specific plasma membrane transporter, which exchanges cytoplasmic Na+ for extracellular K+ is one of these attractive targets for male contraception. Na,K-ATPase ?4 is only expressed in male germ cells of the testis after meiosis and abundant in the sperm flagellum. Importantly, ?4 has functional characteristics that are highly unique and critical for sperm function. Activity of ?4 is essential for maintaining sperm intracellular Na+ levels ([Na+]i) and several vital sperm parameters, including membrane potential (Vm), intracellular Ca+2 ([Ca2+]i) and pH. Importantly, ?4 is crucial for sperm motility and hyperactivation, a key event associated with sperm capacitation. Accordingly, knockout of ?4 in mice results in complete male infertility. From a biochemical standpoint, ?4 has a particularly high affinity for the cardenolide ouabain. We have developed a series of synthetic compounds with steroidal and non-steroidal backbone structure, which, by targeting the ouabain binding site of ?4, selectively inhibit ?4 and affect sperm motility. These results provide strong evidence for the suitability of ?4 as a pharmacological target for the control of male fertility. The key to targeting Na,K-ATPase ?4 is that inhibition of this protein will allow to specifically interfere with sperm function, without affecting spermatogenesis and providing temporary and reversible contraception. In addition, the lack of ?4 in somatic cells reduces the chances that its inactivation will produce side effects. However, before the ?4 inhibitors can be moved forward into their application as male contraceptives, it is necessary that their efficacy, drug-target interaction, biomarkers for their in vitro and vivo specificity, side effects and mechanisms of action are identified and optimized for future clinical use. We will test this here by determining the efficacy and selectivity of action of our inhibitors on Na,K-ATPase ?4 activity, on sperm motility and on sperm parameters that are controlled by Na,K-ATPase ?4 activity, including sperm [Na+]i, [Ca2+]i, pH, Vm, sperm hyperactivation, motility and fertility. Finally, we will determine the pharmacokinetic parameters, safety and contraceptive effectiveness of Na,K-ATPase ?4 inhibitors in mating trials in mice. The rationale for the research is that once the suitability of cardenolides as targets of Na,K-ATPase ?4 is validated, both in vitro and in vivo, they can be advanced as agents for the reversible control of male fertility.