Progesterone (P) has been reported to modulate numerous sperm functions through the binding of P to plasma membrane. One of the effects is an increase in sperm hyperactivation, which is known to be cAMP-dependent. To evaluate the effect of P on cAMP levels, human spermatozoa were incubated 2 h with increasing P concentrations. P significantly induced cAMP increase in a dose-dependent manner, reaching a 3-fold increase at 100 micromol/L (P < 0.01). During the study of the kinetics of P effect, two cAMP peaks were observed: one occurring after a 30-min incubation, with a 1.5-fold increase (P < 0.05), and the second one after a 120-min incubation, with a 2.5-fold increase (P < 0.01). These effects of P on cAMP levels correlated with significant rises in the percentage of hyperactivated spermatozoa, occurring at the same times as those of cAMP. To evaluate the Ca++-dependence of these P effects, the experiments were performed in the presence of and in the absence of Ca++ in the incubation medium. The effects of P at the 30th min and the 120th min were completely abolished in the absence of Ca++. Moreover, calcium ionophore A23187, after a 30-min incubation, induced an increase in cAMP levels identical to that obtained with P. The effect of P was partially reproduced by gamma-amino-butiric acid (GABA) and inhibited by GABA antagonist picrotoxin. It was also inhibited by tyrosine kinase inhibitor genistein but not by RU486. Based on these findings, we conclude that P induces Ca++-dependent cAMP increase in human sperm, that this effect is likely caused by the influx of Ca++ (previously reported), and that the effect partially involves GABA(A)-like receptors.