Phospholipase A₂ (PLA₂) is activated in spermatozoa in response to progesterone and Ca²⁺ ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA₂. We investigated whether PLA₂ is involved in ZP-stimulated acrosomal exocytosis, if Ca²⁺ is required for activation of PLA₂, and signal transduction pathways modulating PLA₂ using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca²⁺ medium with [¹⁴C]choline chloride or [¹⁴C]arachidonic acid and were then exposed to millimolar Ca²⁺ and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca²⁺ and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA₂ activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA₂ inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca²⁺ or in medium with millimolar Ca²⁺ and EGTA or La³⁺ resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin, a G_i protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA₂ plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA₂ activation requires Ca²⁺ internalization, and that PLA₂ activation is regulated by signal transduction pathways involving G proteins and DAG.