We have investigated pathways of lipid metabolism in spermatozoa and generation of various metabolites with potential messenger functions during exocytosis stimulated with A23187/Ca2+. Stimulation of boar spermatozoa resulted in a considerable rapid increase in saturated/unsaturated 1,2-diacylglycerol (1,2-SU-DAG) and, concomitantly, a substantial reduction in disaturated 1,2-diacylglycerol (1,2-DS-DAG), and in phosphatidylcholine (PC). These changes preceded the onset of exocytosis. Phosphatidic acid was sometimes generated in parallel, but usually rose later, suggesting that 1,2-SU-DAG may be formed directly by phospholipase C action. Lipid changes observed in stimulated spermatozoa that have been prelabelled with several lipid precursors ([14C]palmitic acid, [14C]glycerol, [14C]choline, or [14C]arachidonic acid) suggested the existence of a unique process involving the utilization of the high basal levels of 1,2-DS-DAG to form 1,2-SU-DAG, with the latter being subsequently employed to replenish the PC pool. An ensuing generation of lysoPC and arachidonic acid, which paralleled the occurrence of exocytosis, revealed that the newly synthesized PC was hydrolyzed by phospholipase A2. The highest levels of 1,2-SU-DAG, minimum levels of 1,2-DS-DAG, and the regeneration of the PC pool were tightly coupled to the beginning of visible exocytosis. These results suggest that changes in these lipid metabolites may be fundamental processes during acrosomal exocytosis occurring in response to physiological agonists.