Solubilized oocyte zonae pellucidae promoted acrosomal exocytosis in fura-2- or carboxyfluorescein-loaded, mature bovine sperm. Associated elevations of internal [Ca2+] and pH in sperm suspensions were first detectable at 2-5 min, without apparent temporal resolution, and increased monotonically thereafter. Video imaging of fura-2-loaded, single cells identified a responsive subpopulation, destined to undergo exocytosis, that displayed no early transient but manifested lags of 1-7 min then sustained elevations of internal [Ca2+]. Both the zona-induced exocytosis and dye responses were diminished for functionally immature sperm and for mature sperm treated preliminarily with pertussis toxin. Together, these results indicate that a developmentally regulated mechanism of signal transduction employs G protein(s) to couple the physiological (zona) agonist to alterations of the internal ionic mediators of acrosomal exocytosis.