The presence, distribution, and levels of phospholipase A2 and ATPases activities in those structures of the guinea pig spermatozoa that participate in the acrosome reaction were studied, both before and after capacitation, as well as during the acrosome reaction induced in vitro. Spermatozoa were collected from the cauda epididymis and incubated in the absence and presence of 1.15 mmol/L calcium, with and without the addition of 1 mumol/L A23187. Membrane fractions were recovered by vortexing and discontinuous sucrose density gradient centrifugation. Most of the Na+, K(+)-ATPase was recovered in the acrosome-free spermatozoa, but a clear, distinct presence of this enzyme was observed in the plasma membrane (25 against 101 nmoles Pi released per milligram of protein, respectively). The activity of this enzyme in the periacrosomal plasma and in the outer acrosomal membrane increased during calcium incubation. Ca2(+)-dependent ATPase was found in both membrane fractions, being higher in the periacrosomal plasma membrane. The addition of calcium induced a significant inhibition of this acrosomal ATPase, whereas the activity in the acrosome-free spermatozoa increased. The activity of phospholipase A2, under all experimental conditions, was found to be restricted to the soluble fraction.