Isolated neurons of Helix aspersa were dialyzed and voltage clamped under conditions that isolate the Ca current. The rapid time-dependent run-down, or washout, of Ca current could be slowed by addition of 1 mM EGTA to the dialysis solution. A more effective means of slowing washout was the use of agents that promote protein phosphorylation, such as cAMP, Mg-ATP and the catalytic subunit (CS) of cAMP-dependent protein kinase, along with leupeptin, a tripeptide inhibitor of proteases. In the presence of these agents, no internal EGTA was required to prevent Ca current washout. Thus, during dialysis with 100 microM leupeptin, 7 mM Mg-ATP and 20 micrograms/ml CS, the Ca current remained stable for up to several hours. The rate of Ca-dependent inactivation of the current that occurs during a depolarizing step showed only a small decline during prolonged dialysis. Under these conditions, introduction of 10 microM calmodulin plus 40 micrograms/ml calcineurin, a Ca-calmodulin-dependent phosphatase, caused a significant increase in the rate of Ca current inactivation during a depolarizing step. This increase in rate of inactivation, as well as the original inactivation, was eliminated by introduction of EGTA or replacement of external Ca with Ba, results that are consistent with the ion dependency for activation of calcineurin. When internal ATP was replaced with ATP-gamma-S, a hydrolysis-resistant analogue, the rate of Ca current inactivation slowed, providing further evidence that inactivation involves a dephosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)