The small GTP-binding protein Rab3A (identical to smg p25A) is expressed in neural/exocrine/endocrine cells, is distributed between the cytosol and secretory vesicle membranes, and may cycle between these locations to regulate exocytosis. It is proposed that the GTP/GDP state of Rab3A controls this distribution. In PC12 cells, cytosolic Rab3A is predominantly GDP-bound, whereas membrane-associated Rab3A is approximately 50% GTP-bound. Two cytosolic factors, GDP dissociation inhibitor (GDI) and guanine nucleotide releasing factor (GRF), act only on GDP.Rab3A, and preferentially with post-translationally modified Rab3A. Rab3A GTPase-activating protein (GAP) does not preferentially act on processed Rab3A, and interacts selectively with GTP.Rab3A. GDI antagonizes GRF but not GAP activity toward Rab3A. These data are consistent with the concept of an ordered Rab3A cycle controlled by factors that regulate the guanine-nucleotide binding state of Rab3A.