Sperm motility alterations occur in two stages; motility initiation during epididymal transit and transition to hyperactivated motility within the female reproductive tract. We have demonstrated that ejaculated primate sperm contain the serine/threonine PP-1, its endogenous regulators and that treatment of human sperm with the cell-permeable protein phosphatase inhibitor calyculin-a (CL-A) stimulates motility. Our objectives were to determine macaque caput and caudal epididymal sperm PP activity, caput and caudal epididymal sperm motility following CL-A treatment, and PP activity of ejaculated sperm treated with dibutyryl cAMP (dbcAMP) and caffeine which stimulates hyperactivated motility. Protein phosphatase activity was determined using 32P-labeled phosphorylase-` in the absence of divalent cations. Motility analysis was conducted using a computer-assisted motility analysis of system following sperm treatment with 0, 10, 20, 50 or 100 nM CL-A for 10 min. Macaque caput epididymal sperm sonicates contained 4-6-fold more PP activity in comparison to caudal epididymal sperm sonicates. Both caput and caudal epididymal sperm sonicates contained heat-stable PP inhibitor activity as well as Mg-ATP dependent glycogen synthase kinase-3 activity. Treatment of caput epididymal sperm with CL-A increased the percent of motile sperm in a dose-dependent manner from 9% (0 nM CL-A) to 30% (100 nM CL-A) whereas CL-A did not influence the percent of motile caudal epididymal sperm. Treatment of in vitro-capacitated macaque sperm with dbcAMP and caffeine for 1 hr resulted in a significant decrease (P<0.05) in protein phosphatase activity in comparison to nontreated in vitro-capacitated sperm (1.3 q 0.32 and 2.04 q 0.19 x 10-2 nM/min/106 sperm, respectively). These results implicate PP in regulating motility and begin to address mechanisms which control motility initiation of epididymal sperm and the transition to hyperactivated motility.