Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca(2+) ([Ca(2+)]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca(2+)]i before the increase. Both directions of the [Ca(2+)]i change were completely blocked in high K(+) seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca(2+)]i although both cGMP and cAMP increased the [Ca(2+)]i. The decrease in the [Ca(2+)]i induced by cGMP was more notable following a second photolytic event, once [Ca(2+)]i had been elevated by an initial flash. This pattern of [Ca(2+)]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca(2+)]i decrease is due to a Na(+)/Ca(2+) exchanger activity, stimulated by hyperpolarization mediated by K(+) efflux through cGMP-regulated K(+) channels.