We have investigated the production of diacylglycerol (DAG) and phosphatidate (PtdOH) during the exocytosis of the sperm acrosome. Ram spermatozoa treated with Ca2+ and the ionophore A23187 experienced a rapid breakdown of the polyphosphoinositides (PPIs), and a rise in [32P]Pi-labelled PtdOH and DAG mass; PtdOH mass, however, was unaffected. Treatment with Ca2+/A23187 and the DAG kinase inhibitor R59022 resulted in a dose-dependent increase in DAG mass and a concomitant decrease in [32P]PtdOH; such treatment showed a dose-dependent stimulation of acrosomal exocytosis. Pre-incubation with exogenous PtdOHs before stimulation with Ca2+/A23187 did not affect the time-course of exocytosis, whereas treatment with Ca2+/A23187 and exogenous DAGs (dioctanoylglycerol, oleoyl-acetyl-glycerol, or dioleoylglycerol) resulted in a dose-dependent stimulation of acrosomal exocytosis. Our results suggest that DAG, rather than PtdOH, is the important metabolite generated upon PPI hydrolysis; however, since spermatozoa lack protein kinase C, the target of DAG in most cells, a role for DAG in acrosomal exocytosis is as yet unclear.