The functional interaction of the estrogen-induced uterine enzyme glycerylphosphorylcholine (GPC) diesterase with epididymal rat sperm before and after incubation under capacitating conditions was investigated indirectly, by measuring the glycerol phosphate (GP) released on enzymatic hydrolysis of GPC and using oxygen consumption as a measure of oxidative phosphorylation by the sperm. Freshly released, washed sperm metabolized GP but not GPC. Utilization of GP was found to be inhibited by 2 microM oligomycin but was enhanced dose-dependently in the presence of the uncoupling agent, 2,4-dinitrophenol, indicating that the process was essentially similar to that occurring in somatic mitochondria in that it involved electron transport via ubiquinone and the cytochrome system and was coupled to ATP synthesis. However, on incubation of sperm under conditions supporting capacitation, which led to a striking increase in oxygen consumption, the presence of GP decreased oxygen uptake significantly, in a manner similar to that occurring in the presence of the glycolysable substrate, glucose. The implications of these GPC diesterase-induced metabolic alterations in sperm function are discussed.