Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to obtain fertilization of the oocyte in vivo and in vitro. Although most of the biochemical/ biophysical events that occur during capacitation in vitro have been characterized, the molecular mechanisms underlying these complex events are still obscure. Increases of intracellular free Ca2+ concentrations ([Ca2+]i) and protein tyrosine phosphorylation have previously been demonstrated during in vitro capacitation of human spermatozoa. In the present study we investigated the relationship between extracellular/intracellular Ca2+, protein tyrosine phosphorylation, and tyrosine kinase and phosphatase activities during sperm capacitation. We report that the increase in tyrosine phosphorylation of several protein bands that occurs during sperm capacitation is independent of the presence of Ca2+ in the external medium and, at least partially, of the increase in [Ca2+]i occurring during the process. Indeed, the spontaneous increase in phosphorylation was still present in Ca(2+)-free/EGTA-containing-medium and in the presence of the intracellular Ca2+ chelator BAPTA/AM. Moreover, phosphorylation of proteins and protein tyrosine kinase (PTK) activity was enhanced if spermatozoa were incubated in Ca(2+)-free medium, suggesting the presence of Ca(2+)-inhibited tyrosine kinase(s) in human sperm. This hypothesis is further substantiated by the lower tyrosine phosphorylation observed after incubation with the ionophore A23187 and the endoplasmic Ca(2+)-ATPase inhibitor thapsigargin, which promote Ca2+ influx in human sperm. The ability of the cells to undergo acrosome reaction in response to progesterone, which can be considered a functional endpoint of capacitation, was highly compromised when spermatozoa were incubated in Ca(2+)-free medium or in the presence of EGTA, confirming that Ca2+ is required for sperm capacitation. Conversely, in the presence of erbstatin, a inhibitor of tyrosine kinase activity, which blunts tyrosine phosphorylation during capacitation, response to progesterone was maintained, suggesting that tyrosine phosphorylation must be kept at a low level (physiologically by the presence of Ca2+ in the external medium, or pharmacologically by the presence of erbstatin) in order to obtain response to progesterone. This mechanism may be important in vivo during sperm transit in the female genital tract to ensure appropriate timing of full capacitation in the proximity of the oocyte.