Rab3A, Rab3B, Rab3C, and Rab3D constitute a family of GTP-binding proteins that are implicated in regulated exocytosis. Various localizations and distinct functions have been proposed for different and occasionally even for the same Rab3 protein. This is exemplified by studies demonstrating that deletion of Rab3A in knock-out mice results in dysregulation of the final stages of exocytosis, whereas overexpression of Rab3A in neuroendocrine cells causes nearly complete inhibition of Ca(2+)-triggered exocytosis. We have now examined the properties of all Rab3 proteins in the same assays, with the long-term goal of identifying a common conceptual framework for their functions. Using quantitative immunoblotting, we found that all four Rab3 proteins were expressed in brain and endocrine tissues, although at widely different levels. Rab3A, Rab3B, and Rab3C co-localized to synaptic and secretory vesicles consistent with potential redundancy, whereas Rab3D was expressed at high levels only in the endocrine pituitary (where it was more abundant than Rab3A, Rab3B, and Rab3C combined), in exocrine glands, and in adipose tissue. In transfected PC12 cells, all four Rab3 proteins strongly inhibited Ca(2+)-triggered exocytosis. Except for a mutation that fixes Rab3 into a permanently GDP-bound state, all Rab3 mutations tested had no effect on this inhibition, including a mutation in the calmodulin-binding site that was described as inactivating (Coppola, T., Perret-Menoud, V., Lüthi, S., Farnsworth, C. C., Glomset, J. A., and Regazzi, R. (1999) EMBO J. 18, 5885-5891). Unexpectedly, overexpression of wild type Rab3A and permanently GTP-bound mutant Rab3A in PC12 cells caused a loss of secretory vesicles and an increase in constitutive, Ca(2+)-independent exocytosis that correlated with the inhibition of regulated Ca(2+)-triggered exocytosis. Our data indicate that overexpression of Rab3 in PC12 cells impairs the normal control of the final step in exocytosis, thereby converting the regulated secretory pathway into a constitutive pathway. These results offer an hypothesis that reconciles Rab3 transfection and knock-out studies by suggesting that Rab3 functions as a gatekeeper of a late stage in exocytosis.