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Abstract Background: Rab3A, a member of the Rab3 small G protein family, regulates Ca2+ -dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP) and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). We have recently found a novel protein, named rabconnectin-3, which is co-immunoprecipitated with Rab3 GEP or GAP from the extract of the crude synaptic vesicle (CSV) fraction of rat brain. Rabconnectin-3 is abundantly expressed in the brain where it is associated with synaptic vesicles. We have found that two more proteins are co-immunoprecipitated with Rab3 GEP from the CSV fraction of rat brain. We attempted here to isolate and characterize one of them. Results: We determined its partial amino acid sequence, cloned its cDNA from a human cDNA library, and determined its primary structure. The protein consisted of 1490 amino acids (aa) and showed a calculated molecular weight of 163 808. The protein had 7 WD domains. The protein was abundantly expressed in the brain where it co-localized with rabconnectin-3 on synaptic vesicles. The protein formed a stable complex with rabconnectin-3. We named this protein rabconnectin-3β and renamed rabconnectin-3 rabconnectin-3α. Rabconnectin-3β, but not rabconnectin-3α, directly bound Rab3 GEP. Neither rabconnectin-3α nor -3β directly bound Rab3 GAP. Conclusion: These results indicate that rabconnectin-3 consists of the α and β subunits and binds directly Rab3 GEP through the β subunit and indirectly Rab3 GAP through an unidentified molecule(s). [ABSTRACT FROM AUTHOR]