The role of Ca+2 in the acrosome reaction of echinoid and mammalian sperm was investigated using the Ca+2 transporting ionophore A23187. The ionophore induced morphologically normal acrosome reactions in both types of sperm (as assessed by electron microscopic observation of echinoid sperm and phase contrast microscopic observation of mammalian sperm). In echinoids, these reactions were immediate. In the guinea pig and hamster, ionophore significantly decreased the capacitation interval; early reactions were accompanied by activation of motility. Ionophore induced reactions were affected by sperm, ionophore and Ca+2 concentrations. Since both ionophore induced and natural reactions require extracellular Ca+2, it is suggested that an influx of Ca+2 represents the initial step of the acrosome reaction. Under natural conditions, the permeability change which results in Ca+2 influx may be induced in echinoid sperm by egg jelly and may occur in mammalian sperm during capacitation. Ionophore A23187 should prove an experimentally useful drug for further study of the acrosome reaction since its effect on cells is understood, it induces synchronous reactions in a high percentage of sperm, and it conveniently reduces the capacitation interval in mammalian sperm.