Insulin, carboxypeptidase-H (CP-H), and glutamate decarboxylase (GAD) have been identified as potential autoantigens in insulin-dependent diabetes mellitus (IDDM). Previous studies have described immunoreactive insulin as a surface molecule on the plasma membrane of rat islet cells and suggested that cell-surface insulin was derived during exocytosis by the fusion of insulin secretory granules with the β-cell plasma membrane. These findings predict that insulin and other secretory granule-derived proteins such as the putative autoantigen CP-H may be colocalized with insulin at specific sites of exocytosis on the β-cell surface. In studies to test this hypothesis, cell-surface staining of dispersed rat islet cells occurred in a granule-like pattern with antibodies for CP-H and insulin. The specificity of the CP-H antiserum was confirmed by immunoblotting and indicated that the antiserum was essentially monospecific for CP-H. Confocal laser microscopy confirmed that immunoreactive staining for CP-H and insulin was confined to the β-cell surface. Colocalization of CP-H and insulin on the cell surface of β-cells was demonstrated by double staining with antibodies to CP-H and insulin, and the percentage of β-cells positive for both of these autoantigens increased twofold with increases in insulin secretion. In contrast, islet cells failed to reveal cell-surface staining for GAD65, another putative autoantigen in IDDM, under either basal or insulin stimulatory conditions or following exposure of islet cells to the cytokines interleukin-1β, tumor necrosis factor-α, and recombinant human interferon-γ. These results demonstrate that the insulin secretory granule–derived proteins, insulin and CP-H, colocalize on the cell surface of β-cells during exocytosis and in this manner could be recognized by components of the immune system under certain disease settings. These findings further delineate a cellular mechanism whereby the functional activity of the pancreatic (β-cell, i.e., resting versus actively secreting, may correlate with cell-surface localization of antigens and raises the possibility that other unidentified granule derived antigens also may colocalize at sites of exocytosis on the β-cell membrane.