The stimulus-response coupling pathway for glucose-regulated insulin secretion has implicated a rise in cytosolic [Ca2+]i as a key factor to induce insulin exocytosis. However, it is unclear how elevated [Ca2+]i communicates with the pancreatic beta-cell's exocytotic apparatus. As Rab3A is a model protein involved in regulated exocytosis, we have focused on its role in regulating insulin exocytosis. By using a photoactivatable cross-linking synthetic peptide that mimics the effector domain of Rab3A and microsequence analysis, we found calmodulin to be a major Rab3A target effector protein in pancreatic beta-cells. Coimmunoprecipitation analysis from pancreatic islets confirmed a Rab3A-calmodulin interaction in vivo, and that it inversely correlated with insulin exocytosis. Calmodulin affected neither GTPase nor guanine nucleotide exchange activity of Rab3A. The calmodulin-Rab3A interaction was pH- and Ca2+-dependent, and it was preferential for GTP-bound Rab3A. However, Rab3A affinity for calmodulin was relatively low (Kd = 18-22 micromol/l at 10(-5) mol/l [Ca2+]) and competed by other calmodulin-binding proteins that had higher affinity (e.g., Ca2+/calmodulin-dependent protein kinase-2 [CaMK-2] [Kd = 300-400 nmol/l at 10(-5) mol/l [Ca2+]]). Moreover, the Ca2+ dependence of the calmodulin-Rab3A interaction (K0.5 = 15-18 micromol/l [Ca2+], maximal at 100 micromol/l [Ca2+]) was significantly lower compared with that of the calmodulin-CaMK-2 association (K0.5 = 40 micromol/l [Ca2+], maximal at 1 mmol/l [Ca2+]). The data suggested that a transient Rab3A-calmodulin interaction might represent a means of directing calmodulin to the cytoplasmic face of a beta-granule, where it can be subsequently transferred for activation of other beta-granule-associated calmodulin-binding proteins as local [Ca2+]i rises to promote insulin exocytosis.