Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) which stabilizes the Gα(i/o) subunits as an AGS3/Gα(i/o)-GDP complex. It has recently been demonstrated in reconstitution experiments that the AGS3/Gα(i/o)-GDP complex may act as a substrate of resistance to inhibitors of cholinesterase 8A (Ric-8A), a guanine exchange factor (GEF) for heterotrimeric Gα proteins. Since the ability of Ric-8A to activate Gα(i/o) subunits that are bound to AGS3 in a cellular environment has not been confirmed, we thus examined the effect of Ric-8A on cAMP accumulation in HEK293 cells expressing different forms of AGS3 and Gα(i3). Co-immunoprecipitation assays indicate that full-length AGS3 and its N- and C-terminal truncated mutants can interact with Ric-8A in HEK293 cells. Yeast two-hybrid assay further confirmed that Ric-8A can directly bind to AGS3S, a short form of AGS3 which is endogenously expressed in heart. However, Ric-8A failed to facilitate Gα(i)-induced suppression of adenylyl cyclase, suggesting that it may not serve as a GEF for AGS3/Gα(i/o)-GDP complex in a cellular environment.