The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.