Guinea-pig spermatozoa, incubated in a minimal culture medium (MCM-PL) containing 150 mM-Na+ and no added K+, capacitate within 2 h and respond to added Ca2+ with maximal percentage of acrosome reactions. When spermatozoa, initially capacitated in Medium MCM-PL, were washed and resuspended in saline-based media, the motile spermatozoa showed acrosome reactions only in response to added Ca2+ at an extracellular pH greater than 7.4. In contrast, resuspension of capacitated spermatozoa into isotonic sucrose or choline-based media containing no added Na+ and K+ (pH 7.8) did not support acrosome reactions in response to the addition of Ca2+. Examination under the electron microscope showed that these spermatozoa responded to added Ca2+ with swelling or cavitation of the acrosomal matrix but fusion of the plasma membrane and acrosomal membrane did not occur. Treatment of spermatozoa with monensin, a monovalent cationic ionophore, induced rapid acrosome reactions in the presence of extracellular Ca2+ and Na+. In contrast, spermatozoa incubated for 3 h in a sucrose-based Na+-deficient medium or Medium MCM-PL containing 10 mM-KCl failed to give a significant percentage of acrosome reactions in response to the addition of Ca2+, but the addition of ouabain (0.1 mM) prevented the K+ inhibition. Amiloride, a sodium channel blocker in various other tissues, retarded the development of acrosome reactions of spermatozoa incubated in Medium MCM-PL. Based on these data, it appears that an increase in intracellular Na+ has a functional role in acquisition of guinea-pig sperm fertilizing ability.