Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A≫C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K⁺-stimulated secretion of dense core vesicles, indicating that, in β-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxy-terminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.