Ca²⁺-dependent inactivation (CDI) has recently been demonstrated in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca²⁺ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast ( ~ 150 ms) and slow ( ~ 3 s) components in Ba²⁺. Ca²⁺ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 mM to 0.1 mM accelerated slow inactivation, but only in Ca²⁺ as expected from CDI. However, low BAPTA accelerated the fast inactivation in either Ca²⁺ or Ba²⁺, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba²⁺. Increased Ca²⁺, but not Ba²⁺, concentration (5-30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca²⁺ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage-dependence in both Ba²⁺ and Ca²⁺, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.