Abstract Secretory granules maintain a low intragranular pH and it is becoming increasingly recognized that this phenomenon is important to secretion. Cl- entry across the granule membrane is thought to be required to shunt H+ influx via the V-ATPase, thus preventing the build-up of a large transgranular membrane potential. We have shown, for the first time in pancreatic beta cells, that chloride channels (specifically ClC-3) play a role in insulin secretion, likely through regulation of acidification. Previously, we have shown that the CaMKII-gated chloride channel, ClC-3, is functionally expressed in the membrane of insulin-containing granules. Functional studies in isolated beta-cells showed that activation of ClC-3 is permissive for insulin secretion. This is due, at least in part, to the promotion of granular acidification; various strategies to abolish acidification disrupt secretion in a similar manner. These observations are part of a burgeoning literature on the important role played by vesicular ion channels in secretion, as well as the more specific requirement for vesicle acidification in several cases. Recently, we have extended our findings to the ClC-3 knockout mouse. We posit that CaMKII regulates the ClC-3 channel in pancreatic beta-cells and controls granule acidification, rendering granules secretion-competent. Our preliminary data utilizing ClC-3 knock-out mice indicate that beta-cells are defective in exocytosis and the mutant animals exhibit aberrant glucose tolerance. The goal of the present application is to determine the importance of this phenomenon to insulin secretion, understand its mechanism and determine the gating processes that lead to activation of the channel. The present application builds on this foundation and proposes to unravel in molecular detail the role of ClC-3 chloride channels in beta-cell secretion. Acidification of granules may play multiple roles in secretion beyond aiding in the processing of insulin precursors, and it may prove to be a general feature of dense-core granule secretion. In addition to the available drugs that act on K(ATP) channels and increase the triggering signal, novel drugs that would correct a defect in the amplification pathway would be potentially useful in the restoration of adequate insulin secretion in diabetic patients.