We have shown previously that N and Q type channels contribute to the voltage-operated Ca²⁺ current (VOCC) recorded in bovine adrenal chromaffin cells (from the abattoir) and that under prolonged stimulus conditions the exocytotic efficiency of the N-type channel is relatively reduced (Wykes et al 2001). We show that inactivation of the VOCCs in these cells is regulated by Ca²⁺/Calmodulin, with the pharmacologically isolated N type channel displaying the most sensitivity.Inactivation was investigated using a train of depolarisations (50 x 10ms, from 80mV to +20mV at 20 Hz). Fractional inactivation was determined by normalising the peak amplitude of a current to that of the 1^st current in the train. Results presented are the mean inactivation and s.e.m at pulse 50. Perforated patch recordings with extracellular Ca²⁺ produced inactivation of 0.48 +/- 0.02 (n=7), equimolar replacement with Ba²⁺ resulted in less inactivation, 0.86 +/0.03 (n=3). In whole cell experiments increasing the concentration of BAPTA in the electrode solution from 0.3mM to 10mM changed inactivation from 0.42 +/- 0.03 (n=7) to 0.74 +/- 0.03 (n=5). Channels were pharmacologically isolated to assess whether both subtypes possessed the same degree of inactivation. Inactivation of ω-CgTX GVIA (1µM) treated cells was 0.66+/- 0.05 (n=4), whereas for ω-Aga IVA (300nM) treated cells it was 0.32 +/- 0.02 (n=4). The difference in the pharmacologically isolated channel types was significant in an un-paired students t-test, p = 0.0006. Switching from Ca²⁺ to equimolar Ba²⁺ reduced the amount of inactivation observed for the N-type channel from 0.32 +/- 0.02 (n=4) to 0.89 +/- 0.04 (n=2).The molecular mechanisms underlying this effect were investigated. Inhibiting calcinuerin by 20 mins preincubation with 1µM cyclosporine A or by introducing 30µM calmodulin (CaM) inhibitory peptides through the patch pipette did not significantly reduce the level of inactivation. In contrast, adenoviral mediated expression of a mutant CaM deficient in Ca²⁺ binding (Alseikhan, B. A et al 2002) significantly reduced inactivation. In WT CaM expressing cells the inactivation was 0.39 +/- 0.02 (n=13), whereas inactivation in mutant CaM expressing cells was 0.64 +/- 0.03 (n=16) (P<0.05).Results are consistent with CaM acting directly to control N-type channel inactivation in adrenal chromaffin cells and could account for the reduced exocytotic efficiency observed with these channels during intense stimulation.