Testis- and sperm-specific protein phosphatase, PP1γ2, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP1γ2 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP1γ2 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP1γ2 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936–1942). The objectives of this study were to determine: 1) stoichiometry of PP1γ2-sds22 binding and 2) whether PP1γ2 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP1γ2 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP1γ2 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP1γ2 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP1γ2 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP1γ2-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP1γ2 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP1γ2 (39 kDa), suggesting a 1:1 complex between these two proteins. PP1γ2 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP1γ2. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP1γ2 during epididymal sperm maturation.