We have previously reported that calmodulin (CaM) acts directly to control N-type calcium channel inactivation in cultured bovine adrenal chromaffin cells (Wykes & Seward, 2004). Here we report that CaM additionally interacts with the secretory machinery to regulate exocytosis. Stimulus-secretion coupling was examined in single cells using combined measurements of membrane capacitance (ΔCm) and voltage-clamp recording of calcium currents. Cells were clamped at -80 mV in either the perforated patch or whole-cell configuration and Ca²⁺ dependent exocytosis evoked by single voltage steps to +20 mV or a train of depolarisations (10-100ms duration); exocytotic efficiency was derived by dividing ΔCm by the integral of the calcium current. Adenoviral-mediated expression of a mutant CaM incapable of binding calcium (CaM1234) significantly reduced the exocytotic efficiency of brief depolarisations (100 ms) to 0.30 ± 0.03, n=7, from 0.75 ± 0.16, n=5 (p = 0.0008, Student’s t test) in cells overexpressing wild type CaM (CaM_wt). The exocytotic efficiency to longer depolarisations (≥ 200ms) however was not significantly different between cells expressing CaM₁₂₃₄ and CaM_wt. This suggests that Ca²⁺-CaM is required for filling and/or release from a rapidly releasable pool of vesicles which is easily depleted, but not from the slowly releasable pool which dominates exocytotic responses measured with prolonged responses. A protocol designed to look at different releasable pools of vesicles (a train of 6, 10ms pulses, followed by 4, 100ms pulses to +20 mV) confirmed this observation. With this protocol, the 10ms stimuli will result in a ΔCm that reflects fusion from the immediately releasable vesicle pool, while the further 4 longer pulses result in a second bout of secretion dominated by release from the slowly releasable vesicle pool (Voets, 2000). CaM1234 expression reduced the size of the immediately releasable vesicle pool, quantified by measuring responses to a pair of 10 ms depolarisations, to 14 ± 5 fF, n=18, corresponding to ~ 7 vesicles compared with 58 ± 17 fF, n = 15 or 29 vesicles in cells overexpressing CaM_wt, p = 0.01. Taken together, these results place CaM as a central molecule that controls Ca²⁺ entry (Ca²⁺ channels) and the exocytotic and endocytotic machinery to regulate stimulus-coupled secretion. Primary cultures of bovine chromaffin cells were made weekly from glands collected from a local abattoir.