The effects of proteolytic enzymes on ionic conductances of squid axon membranes have been studied by means of the voltage clamp technique. When perfused internally alpha-chymotrypsin (1 mg/ml) increased and prolonged the depolarizing after-potential. Sodium inactivation was partially inhibited causing a prolonged sodium current, and peak sodium and steady-state potassium currents were suppressed. The time for sodium current to reach its peak was not affected. Leakage conductance increased later. On the other hand, carboxypeptidases A and B, both at 1mg/ml, suppressed the sodium and potassium conductance increases with little or no change in sodium inactivation. The mechanism that controls sodium inactivation appears to be associated with the structure of membrane proteins which is modified by alpha-chymotrypsin but not by carboxypeptidases and is located in a position accessible to alpha-chymotrypsin only from inside the membrane.