The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic beta-cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin-O-permeabilized MIN6 cells was inhibited by anti-annexin XI antibody, when the release was stimulated by either Ca2+ or GTP-gammaS, but not by a protein kinase C-activating phorbol ester. Inhibition of insulin release by anti-annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic beta-cells.