The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic -cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the -cell line INS-1E wild type Noc2, Noc2^65E and Noc2^58A, a mutant capable of interacting with Rab27 but not Rab3, colocalised with insulin-containing vesicles. In contrast, two mutants (Noc2^138S,141S and Noc2^{154A,155A,156A}) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild type Noc2, Noc2^65E or Noc2^58A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic -cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13.