Sea urchin sperm contain two isozymes of creatine kinase (CrK) in the sperm head and tail, as termini of a phosphocreatine shuttle to transport energy. The head isozyme is located at the mitochondrion. By using an antibody prepared against denatured flagellar CrK, we now show that the tail isozyme exists along the entire flagellum. This unusual CrK isozyme, of Mr 145 kDa, is a component of the flagellar axoneme as indicated by electron microscopic immunolocalization and cell fractionation. Flagellar CrK specifically reassociated with extracted sperm axonemes as well as with in vitro polymerized sea urchin egg microtubules. Neither sperm mitochondrial CrK nor mammalian muscle CrK bound to axonemes under similar conditions. Thus, although the two sperm isozymes have similar kinetic properties, they differ in affinity for microtubules, a characteristic that may determine the regional differentiation needed for establishing a phosphocreatine shuttle.