Sperm motility initiation, capacitation, and hyperactivation are modulated by an interplay of intracellular Ca2+, cAMP, and pH. Mechanisms by which these mediators alter sperm function have not been elucidated but may involve reversible alterations in regulatory protein phosphorylation. Studies were designed 1) to investigate the influence of the protein phosphatase (PP) inhibitor calyculin A (CA) on human sperm motility and 2) to characterize the CA-sensitive PP and its endogenous regulators in human and rhesus monkey sperm. CA (50 nM) treatment of human sperm resulted in an increase in percentage motility and an acceleration in mean path velocity. Inhibition of either protein phosphatase-1 (PP1) or protein phosphatase-2A (PP2A) could be responsible for this motility stimulation, since both of these phosphatases are sensitive to nanomolar quantities of CA. PP activity in human (n = 4) and rhesus monkey (n = 4) sperm sonicates was measured using [32P]-phosphorylase-a, the preferred substrate for PP1 and PP2A, in the absence of divalent cations. Human (6.2 +/- 4.5 x 10(-2) mU/10(6) sperm) and monkey (4.3 +/- 0.8 x 10(-2) mU/10(6) sperm) sonicates contained activity tentatively identified as PP1 on the basis of inhibition profiles in okadaic acid (OA) and CA. Western blot analysis with antibodies against various isoforms of PP1 subsequently documented the presence of PP1 gamma 2 in human and monkey sperm. PP1 activity in most tissues is regulated by the heat-stable inhibitors I1 or I2. Sperm sonicates contained inhibitor activity similar to I2 as well as glycogen synthase kinase-3 (GSK-3) activity, which is involved in the activation of the PP1-I2 complex. These results indicate, for the first time, that human and rhesus monkey sperm contain PP1 and regulators of PP1 and that inhibition of PP1 activity by CA can enhance motility.