The presence of ATP in the genital tract fluid of mammals provokes questions regarding its function in the fertilization process. We investigated the effect of extracellular ATP (ATPe) on the activation of bovine spermatozoa. A signal transduction mechanism for ATP involving the receptor-mediated release of second messengers is described. Treatment of spermatozoa with ATP, uridine triphosphate (UTP), or 2-methylthio-ATP resulted in a concentration-dependent increase of acrosomal exocytosis, whereas treatment with either AMP or adenosine induced little exocytosis. This suggested that the receptor involved is of the P₂ and not the P₁ type. Several lines of evidence also suggest that the ATP purinoceptor is of the P_2y and not the P_2x type. First, the acrosome reaction was induced by the P_2y-agonists ATP, UTP, or 2-methylthio-ATP, but no effects were shown by the P_2x-agonists α,β-methylene-ATP or β,γ-methylene-ATP. Second, ATP-induced acrosomal exocytosis was inhibited by the P_2y antagonists, but not by the P_2x antagonists. Third, enhanced Ca² uptake into the cells was observed with ATP and 2-methylthio-ATP, but not with β,γ-methylene-ATP. Additionally, ATP induced elevation of intracellular Ca² and cAMP, and the effect on cAMP was predominantly enhanced by including Ca² and the Ca² -ionophore A23187 in the incubation medium. Extracellular ATP also activates protein kinase Cα (PKCα), and the acrosome reaction, stimulated by ATPe, is inhibited by a PKC-specific inhibitor. In summary, we suggest that ATPe activates the P₂ purinoceptor that elevates [Ca² ]_i, which leads to PKCα activation and culminates in acrosomal exocytosis.