The enzyme PP1γ2 is a testis- and sperm-specific isoform of type 1 protein phosphatase (PP1), and it is the only isoform of PP1 in spermatozoa. The enzyme PP1γ2 is essential for spermatogenesis and is also a key enzyme in the development and regulation of sperm motility. The carboxy terminus of the enzyme contains a consensus amino acid sequence for phosphorylation by cyclin-dependent kinases. Using antibodies specific to this phosphorylated amino acid sequence domain, we found that phosphorylated PP1γ2 is present in bovine epididymal spermatozoa. The level of phosphorylated PP1γ2 is significantly higher in motile caudal compared to immotile caput epididymal spermatozoa. A number of treatments, such as 2-chloro adenosine, cAMP analogues, cAMP phosphodiesterase inhibitors, and calcium, which stimulate sperm motility, did not alter the level of phosphorylated PP1γ2. However, calyculin A, which is an inhibitor of protein phosphatase subtypes PP1 and PP2A, significantly increases the level of phosphorylated PP1γ2 in both caput and caudal epididymal spermatozoa. Partial purification by column chromatography showed that phosphorylated PP1γ2 is catalytically active. Phosphorylated PP1γ2 is the only spontaneously catalytically active form of the enzyme in caudal sperm extracts. Western blot analysis shows that the enzyme cyclin-dependent kinase 2, one of the enzymes that phosphorylates the consensus domain at the carboxy terminus in PP1 isoforms, is present in spermatozoa. Western blot analysis of proteins extracted from purified head and tail fragments of spermatozoa showed that phosphorylated PP1γ2 is present predominantly in the sperm head. Fluorescence immunocytochemistry also showed that phosphorylated PP1γ2 is present predominantly in the posterior region of the sperm head. The distinct subcellular localization and changes in its level during sperm maturation suggest a possible role for sperm phosphorylated PP1γ2 in signaling events during fertilization.