Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Gα subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Gα·GDP subunits and forms a stable nucleotide-free Ric-8A:Gα complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Gα subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Gα·GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Gαi1 and that of Ric-8A with the AGS3-C:Gαi1·GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Gαi1·GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Gαi1·GDP. Subsequent dissociation of AGS3-C and GDP fromGαi1 yields a stable nucleotide free Ric-8A·Gαi1 complex that, in the presence of GTP, dissociates to yield Ric-8A andGαi1· GTP. AGS3-C does not induce dissociation of the Ric- 8A·Gαi1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Gαi1·GDP ensures unidirectional activation of Gα subunits that cannot be reversed by AGS3.