Effect of cytoplasmic chloride ion concentration ([Cl^-]_i) on secretory activity was studied in mouse adrenal slices. Ca²⁺-dependent exocytosis was triggered by slow photo-release of caged Ca²⁺ (NP-EGTA). Resulting exocytotic burst was measured as membrane capacitance changes (C_m) in whole-cell patch-clamp configuration. Ramp-like changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i, Fura-6F photometry) enabled high temporal resolution and more precise determination of Ca²⁺ dependency of the elicited exocytotic bursts. Increasing [Cl^-]_i to 155 mM resulted in a significant change in the amplitude as well as maximal rate of C_m change. At 155 mM [Cl^-]_i, the amplitude of the secretory bursts was approximately two-times larger compared to low [Cl^-]_i (14 mM). Similarly, the maximal rate of C_m change during bursts was significantly faster at high [Cl^-]_i (400 ± 44 fF/s) compared to the low [Cl^-]_i (233 ± 38 fF/s). Importantly, the sensitivity of the exocytotic machinery did not depend on [Cl^-]_i as Ca²⁺ concentration required for the half maximal exocytotic activity was comparable (EC₅₀ = 0.4 mM). This suggests that [Cl^-]_i modulated secretory vesicle availability rather than their sensitivity for Ca²⁺. Intracellular application of DIDS (disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate) that presumambly blocked chloride channels mimicked the effect of decreased [Cl^-]_i. Taken together, the influence of [Cl^-]_i on secretory amplitude and rate but not on its sensitivity for Ca²⁺ corroborates the modulatory role of chloride ions in the promotion of vesicular maturation.