P/Q-type (Ca_V2.1) and N-type (Ca_V2.2) Ca²⁺ channels are critical to stimulus-secretion coupling in the nervous system; feedback regulation of these channels by Ca²⁺ is therefore predicted to profoundly influence neurotransmission. Here we report divergent regulation of Ca²⁺-dependent inactivation (CDI) of native N- and P/Q-type Ca²⁺ channels by calmodulin (CaM) in adult chromaffin cells. Robust CDI of N-type channels was observed in response to prolonged step depolarizations, as well as repetitive stimulation with either brief step depolarizations or action potential-like voltage stimuli. Adenoviral expression of Ca²⁺-insensitive calmodulin mutants eliminated CDI of N-type channels. This is the first demonstration of CaM-dependent CDI of a native N-type channel. CDI of P/Q-type channels was by comparison modest and insensitive to expression of CaM mutants. Cloning of the C terminus of the Ca_V2.1 α1 subunit from chromaffin cells revealed multiple splice variants lacking structural motifs required for CaM-dependent CDI. The physiological relevance of CDI on stimulus-coupled exocytosis was revealed by combining perforated-patch voltage-clamp recordings of pharmacologically isolated Ca²⁺ currents with membrane capacitance measurements of exocytosis. Increasing stimulus intensity to invoke CDI resulted in a significant decrease in the exocytotic efficiency of N-type channels compared with P/Q-type channels. Our results reveal unexpected diversity in CaM regulation of native Ca_V2 channels and suggest that the ability of individual Ca²⁺ channel subtypes to undergo CDI may be tailored by alternative splicing to meet the specific requirements of a particular cellular function.