The mitochondrion of sea urchin sperm is located at the base of the sperm head, and the flagellum extends from the mitochondrion for approximately 40 microM. These sperm have two known flagellar, non-mitochondrial, enzymatic systems to rephosphorylate ADP. The first involves the phosphocreatine shuttle, where flagellar creatine kinase (Sp-CK) uses phosphocreatine to rephosphorylate ADP. The second system, studied in this report, is adenylate kinase (Sp-AK), which uses 2 ADP to make ATP + AMP. Cloning of Sp-AK shows that, like Sp-CK, Sp-AK has three catalytic domains. Sp-AK localizes along the entire flagellum, and most of it is tightly bound to the axoneme. Sp-AK activity and flagellar motility were studied using demembranated sperm. The specific Sp-AK inhibitor Ap5A blocks enzyme activity with an IC50 of 0.41 microM. In 1 mm ADP, flagella reactivate motility in 5 min; 1 microM Ap5A completely inhibits this reactivation. No inhibition of motility occurs in Ap5A when 1 mm ATP is added to the reactivation buffer. The pH optimum for Sp-AK is 7.7, an internal pH at which sperm are fully motile. The pH optimum for Sp-CK is 6.7, an internal pH at which sperm are immotile. In isolated, detergent-permeabilized flagella, assayed at pH 7.6, the Km for Sp-AK is 0.32 mm and the Vmax is 2.80 microM ATP formed/min/mg of protein. When assayed at pH 7.6, the Sp-CK Km is 0.25 mm and the Vmax 5.25. At the measured in vivo concentrations of ADP of 114 microM, at pH 7.6, the axonemal Sp-AK could contribute approximately 31%, and Sp-CK 69%, of the total non-mitochondrial ATP synthesis associated with the demembranated axoneme. Thus, Sp-AK could contribute substantially to ATP synthesis utilized for motility. Alternatively, Sp-AK could function in the removal of ADP, which is a potent inhibitor of dynein ATPase.