An increase in the concentration of intracellular free Ca² and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca² concentration through modulation of Ca² -ATPase activity or the phosphotyrosine content is under Ca² regulation was investigated using Ca² -ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca² -ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca² concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca² -ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca² concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca² -dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca² concentration. Based on these findings, the present study suggests that Ca² -ATPases are involved in the filling of internal Ca² stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca² , which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.