Mammalian spermatozoa require extracellular Ca2+, some of which must be internalized, to undergo capacitation and acrosomal exocytosis. The mechanisms controlling the intracellular Ca2+ concentration are unclear, but current evidence suggests that a Ca2+-ATPase may be involved. Using treatments that potentially modulate enzyme activity, we investigated this possibility in human spermatozoa; the capacitation state and acrosomal integrity were monitored by chlortetracycline fluorescence. Incubation of cells in the presence of quercetin, a Ca2+-ATPase inhibitor, significantly accelerated the transition from the uncapacitated F pattern of chlortetracycline fluorescence to the capacitated, acrosome-intact B pattern within 1 h. This was followed by an increase in the number of cells displaying the capacitated, acrosome-reacted AR pattern. Since most Ca2+-ATPases in somatic cells are sensitive to calmodulin, we also investigated the effect of the calmodulin antagonist W-7 on chlortetracycline patterns. At 1–125 μmol l−1, W-7 significantly stimulated capacitation and acrosomal exocytosis. Furthermore, W-7 at 1 μmol l−1 proved to be more effective than W-5, a less potent antagonist, suggesting that the observed responses in human spermatozoa did reflect a calmodulin-sensitive mechanism. When the glucose concentration in the culture medium was varied (from 0 to 5.56 mmol l−1) to alter the availability of ATP for enzyme activity, it was found that a reduced concentration of glucose promoted capacitation more rapidly than did the standard concentration of 5.56 mmol glucose l−1. However, maximal changes, particularly in promoting the shift from the B to the AR pattern of chlortetracycline fluorescence, required millimolar concentrations of glucose during the last few minutes before assessment. Finally, the addition of partially purified mouse sperm decapacitation factor (proposed to activate a Ca2+-ATPase and thus maintain a low intracellular Ca2+ concentration) to capacitated human sperm suspensions caused a significant reversal in the capacitation state of cells (from the B to the F pattern). The F pattern of chlortetracycline fluorescence predominates in conditions favouring low concentrations of intracellular Ca2+. From these results, we suggest that a Ca2+-ATPase may play an important role during human sperm capacitation. A time-dependent decrease in endogenous enzyme activity would allow the intracellular concentration of Ca2+ to rise to a critical value necessary for initiation acrosomal exocytosis and subsequent successful fertilization.