The present study explores the mechanism underlying the action of L-arginine on the metabolic activity of spermatozoa. Goat epididymal spermatozoa were incubated with different concentrations of L-arginine to determine its effect on the utilization of glucose, fructose, and pyruvate. NMR techniques have been applied to elucidate the effect of L-arginine, L-lysine, and L-ornithine on the glycolysis of epididymal goat spermatozoa. Whereas 31P NMR has been used to estimate the change of pH in the presence of different concentrations of L-arginine, 13C NMR has been used to estimate the substrate consumption and lactate production. At optimal concentration of L-arginine, the forward metabolic rates have been found to increase by two to three times over control experiments. Arginine is not consumed in these reactions, but acts as an activator. Longitudinal relaxation time (T1) measurements indicate that the guanidino group of L-arginine plays an active role in binding to cells. The amino acid L-lysine is less effective, and L-ornithine is ineffective.