A possible role for protein kinases in the regulation of free cytosolic Ca2+ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca2+ (monitored with the Ca(2+)-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K+. The ability of various drugs to inhibit the cytosolic Ca2+ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the increase in cytosolic Ca2+ induced by high K+ depolarization, as was sphingosine, a drug that inhibits protein kinase C by binding to the regulatory site, but which also inhibits calcium/calmodulin kinase. On the other hand, drugs that inhibit protein kinases by binding to the catalytic site, such as H-7 (1 mM), staurosporine (1 microM), and K252a (1 microM), were ineffective. Activation of protein kinase C, which is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca2+ in depolarized synaptosomes. All of the drugs, including sphingosine, that functionally inhibit the depolarization-induced elevation in cytosolic Ca2+ have in common the ability to bind to calmodulin. Because the drugs that inhibit protein kinases by competing with ATP binding at the active catalytic site did not block the response in this system, we suggest that a calmodulin or a calmodulin-like binding site participates in the regulation of Ca2+ increases after depolarization.