The shape of the concentration-response curve and the amplitude of maximal motility increase for the stimulatory effect of caffeine on human sperm motility were similar to those of EGTA, a calcium chelator, and lanthanum chloride, a calcium entry blocker. On the other hand, the maximal motility stimulation induced by dibutyryl cyclic AMP was similar to that induced by trifluoperazine, a calmodulin antagonist. The antagonism of A23187 on the motility-stimulating effect of EGTA and caffeine was competitive in nature, while that on cyclic AMP was non-competitive. We proposed that there could be two mechanisms for caffeine to stimulate sperm motility: at higher concentrations, caffeine modified calcium translocation; at lower concentrations, caffeine increased cyclic AMP level. The importance of calcium in the regulation of human sperm motility was reconfirmed in this study.