The serine/threonine phosphatase (PP1) isoform PP1 gamma 2, predominantly expressed in the testis, is a key enzyme in spermatozoa. High PP1 gamma 2 catalytic activity holds motility in check in immature spermatozoa. Inhibition of PP1 gamma 2 causes motility initiation in immature spermatozoa and motility stimulation and changes in flagellar beat parameters in mature spermatozoa. The PP1 gamma 2 isoform is present in all mammalian spermatozoa studied: mouse, rat, hamster, bovine, non-human primate and man. We have now identified at least four of its regulatory proteins that regulate distinct pools of PP1 gamma 2 within spermatozoa. Our studies provide new insights into biochemical mechanisms underlying development and regulation of sperm motility. We hypothesize that changes in sperm PP1 gamma 2 activity as a result of phosphorylation and reversible binding of the regulatory proteins to the catalytic subunit are critical in the development and regulation of motility and the ability of sperm to fertilize eggs. Targeted disruption of the Ppp1cc gene, which encodes the PP1 gamma 1 or PP1 gamma 2 isoforms, causes male infertility in mice as a result of impaired spermiogenesis. Our observations suggest that, in addition to motility, the protein phosphatase PP1 gamma 2 might play an isoform-specific function in the development of specialized flagellar structures of mammalian spermatozoa.