The HCO3(-) anion activates sperm motility, an important early step in capacitation, by increasing flagellar beat frequency through a pathway that requires the atypical adenylyl cyclase SACY and the sperm-specific C alpha2 catalytic subunit of PKA. Here we show that the accelerating action of HCO3(-) also requires the continued presence of external Ca2+ (EC50 approximately 0.5 mM), and find that Ca2+ can be replaced by Sr2+ but not by Mn2+. Ca2+ is required for HCO3(-) to elevate cAMP, but not for cAMP-AM to increase beat frequency, indicating that external Ca2+ acts before rather than after stimulation of SACY by HCO3(-). With external Ca2+ present, HCO3(-) does not alter cytosolic or near-membrane [Ca2+]. Removal of external Ca2+ initiates a slow decline in intracellular [Ca2+] and rapid block of the HCO3(-)-evoked acceleration that is not relieved upon increasing internal [Ca2+] by rapid photolysis of caged Ca2+. We also find that the rapid (t(1/2) approximately 10 s) accelerating action of HCO3(-) is slowed more than three-fold by the carbonic anhydrase inhibitor acetazolamide. It is unaltered by the broad spectrum anion transport inhibitor SITS, and is not accompanied by detectable changes in intracellular pH. We propose that external Ca2+ binds an unidentified extracellular protein that is required for HCO3(-) to engage cAMP-mediated activation of motility.