The CLC family is a large family with nine members which fulfill diverse functions, at the plasma membrane or in intracellular compartments. ClC-3 and ClC-5 belong to a sub-branch of intracellular Cl-/H+ antiporters. The physiological relevance of ClC-3 in the central nervous system (CNS) is demonstrated by the Clcn3-/- knock-out animal model, which exhibits a pronounced hippocampal and retinal degeneration[1] as well as alterations in the excitatory/inhibitory neurotransmission [1],[2],[3]. Beside its role in neuronal transmission, ClC-3 was more recently proposed as regulator in the neuroendocrinal system[4],[5]. ClC-5 is required for endocytosis in the proximal tubule, and so far no role has been assigned in neuronal or neuroendocrinal cells. The aim of this project is to characterize the role of ClC-3 in regulated exocytosis of large dense core vesicles (LDCVs) in neuroendocrinal cells. We characterized exocytosis in adrenal chromaffin cells through a combined technique of cell membrane capacitance and amperometric recordings. In contrast to previous results[4],[5] where exocytosis in adult (P60) Clcn3-/- chromaffin cells was severely affected, we could not observe any dramatic effect in exocytosis measured in new born (P0) Clcn3-/- mice. Therefore, we investigated whether another chloride/proton exchanger might compensate for the absence of ClC-3 in early developmental stages. Quantitative real-time PCR experiments showed that ClC-5 is less abundant (down-regulated) in adrenal glands extracted from adult mice. ClC-5 was up-regulated in absence of ClC-3 in new born mice, suggesting a possible compensating role. To test the effect of ClC-5 in exocytosis, we reduced the expression of ClC-5 (via shRNA Knockdown strategy, using the lentiviral expression system) in chromaffin cells isolated from Clcn3-/- P0 mice. The exocytotic response in such double mutant (Clcn3-/-/Clcn5KD) cells was significantly decreased, similarly to the impairment observed in exocytosis measured in Clcn3-/- P60 cells. Furthermore, analysis of single amperometric events did not reveal any impairment in the amount of released catecholamines, indicating that most probably ClC-3 and ClC-5 do not regulate neurotransmitter loading in LDCVs. The reduced spike frequency observed in Clcn3-/- P60 cells and DKOs P0 cells reveals a decreased LDCVs fusion frequency, reflecting a reduced number of primed vesicles. We therefore monitored cell membrane capacitance in response to depolarizing stimuli in order to evaluate the vesicle priming process. (Clcn3-/-/Clcn5KD) cells exhibit a significant reduction in the total cell membrane capacitance as well as in the first 4th depolarizing step, suggesting that Cl-/H+ exchangers regulate LDCVs priming. We conclude that ClC-3 and ClC-5 are crucial regulators in the priming process of LDCVs in adrenal chromaffin cells.