Ca2+ channel inactivation was investigated in neurohypophysial nerve terminals by using patch-clamp techniques. The contribution of intracellular Ca2+ to inactivation was evaluated by replacing Ca2+ with Ba2+ or by including BAPTA in the internal recording solution. Ca2+ channel inactivation during depolarizing pulses was primarily voltage-dependent. A contribution of intracellular Ca2+ was revealed by comparing steady-state inactivation of Ca2+ channels with Ca2+ current and with intracellular [Ca2+]. However, this contribution was small compared to that of voltage. In contrast to voltage-gated Ca2+ channels in other preparations, in the neurohypophysis Ba2+ substitution or intracellular BAPTA increased the speed of inactivation while reducing the steady-state level of inactivation. Ca2+ channel recovery from inactivation was studied by using a paired-pulse protocol. The rate of Ca2+ channel recovery from inactivation at negative potentials was increased dramatically by Ba2+ substitution or intracellular BAPTA, indicating that intracellular Ca2+ inhibits recovery. Stimulation with trains of brief pulses designed to mimic physiological bursts of electrical activity showed that Ca2+ channel inactivation was much greater with 20 Hz trains than with 14 Hz trains. Inactivation induced by 20 Hz trains was reduced by intracellular BAPTA, suggesting an important role for Ca2+-dependent inactivation during physiologically relevant forms of electrical activity. Inhibitors of calmodulin and calcineurin had no effect on Ca2+ channel inactivation, arguing against a mechanism of inactivation involving these Ca2+-dependent proteins. The inactivation behavior described here, in which voltage effects on Ca2+ channel inactivation predominate at positive potentials and Ca2+ effects predominate at negative potentials, may be relevant to the regulation of neuropeptide release.