Calcium tail currents, recorded at -40 mV after repolarization from 7 or 10 ms voltage-clamp depolarizations in axotomized Aplysia neurones in the presence of tetrodotoxin and tetraethylammonium, were used to investigate the inactivation of the calcium conductance without interference from contaminating potassium currents. Prior depolarization with a prepulse (V1) resulted in a reduction in size of the tail currents recorded following the test pulse (V2). The reduction occurred in both the fast (tau 1 less than 0.4 ms) and slow (tau 2 approximately equal to 2.0 ms) components of the tail current. The degree of inactivation remained constant when tail currents were measured at potentials ranging up to 30 mV on either side of the potassium equilibrium potential. Thus, any changes in potassium current must have contributed virtually nothing to the changes in tail current amplitude seen following presentation of the prepulse. Inactivation was greatest following prepulses to potentials (+10 to +40 mV) that produce maximal entry of calcium ions, and declined to about zero as the prepulse approached the calcium equilibrium potential. For V1 potentials above +50 mV, the prepulse caused an apparent short-term facilitation of V2 tail currents in EGTA-injected neurones. This effect, detected up to 50 ms following the pulse, is of uncertain origin. Pressure injection of calcium ions caused reduction in the size of the tail current, which was restored by subsequent injection of EGTA. Tail current amplitude was reduced by presentation of the prepulse for all test pulse voltages, but the measured inactivation declined exponentially towards a minimum with test pulses of increasingly positive potential. Removal of inactivation following a 200 ms prepulse was greatly accelerated by injection of EGTA. The EGTA-resistant inactivation remaining at short times decayed with a time constant of about 0.12 s. The relation of tail current reduction to prepulse voltage is consistent with the interpretation that the EGTA-resistant inactivation remaining at short times depends on entry of calcium ions during the prepulse, as does the EGTA-sensitive inactivation remaining at later times. It is proposed that the 'EGTA-resistant' phase of inactivation results from loading of EGTA with calcium ions near the inner surface of the membrane during sustained calcium entry, allowing the intracellular calcium concentration to rise. The results provide further evidence for a calcium-mediated inactivation of the calcium conductance.(ABSTRACT TRUNCATED AT 400 WORDS)