The protein phosphatase PP1γ2 is critical in the regulation of sperm motility and fertility. Its activity is regulated by its binding proteins and by phosphorylation. We have recently shown that PP1γ2 is phosphorylated and that the amount of phosphorylated PP1γ2 increases during sperm epididymal maturation (Huang et al., Biol Reprod 2004; 70:439–447). Microsequencing revealed that protein 14-3-3 coeluted with phosphorylated PP1γ2 during column chromatography of bovine sperm extracts. Western blot analyses confirmed the presence of protein 14-3-3 not only in bovine spermatozoa but also in spermatozoa of diverse species—bull, hamster, horseshoe crab, monkey, rat, turkey, and Xenopus. The binding between PP1γ2 and protein 14-3-3 was confirmed by coimmunoprecipitation experiments and in pull-down assays with recombinant GST-14-3-3. Western blot analysis and protein 14-3-3 immunoprecipitates with antibodies against the consensus binding domain of protein 14-3-3 reveal that, in addition to PP1γ2, at least two other protein 14-3-3 binding partners are present in spermatozoa. Fluorescence immunocytochemistry results indicate that phosphorylated PP1γ2 and protein 14-3-3 both localize to the postacrosomal region of the head and principal piece of bovine spermatozoa. Together, these results provide conclusive evidence that protein 14-3-3 is present in mature spermatozoa and that PP1γ2 is one of its binding partners.