G protein activated K⁺ channels (GIRK, Kir3) mediate postsynaptic inhibitory effects of neurotransmitters by direct binding of Gbg following activation of G_i/o proteins via numerous G protein coupled receptors. Using Xenopus oocytes, we demonstrate that Ga_i^GDPbg actively regulates GIRK gating. Two Ga_i3 mutants were utilized: "constitutively active" Ga_i3Q204L ("QL"; poor GTPase) and "constitutively inactive" Ga_i3G203A ("GA") which forms a stable complex with Gbg. GIRK was activated by coexpression of Gbg (whole-cell experiments) or by application of purified Gbg to excised membrane patches. Ga_i3GA, with clear difference from other Gbg scavengers, mimicked the wild-type Ga_i3 in reducing the basal current and priming GIRK for activation by Gbg. Ga_i3QL elicited no effect. In in vitro protein interaction studies, purified Gbg enhanced the binding of the whole cytosolic domain of GIRK1 to Ga_i3^GDP or Ga_i3^GTP. This enhancement was not observed with GIRK2. In addition, using two-electrode voltage clamp, we tested Ga and Gbg regulation of homotetramers of GIRK1 and GIRK2. While GIRK2 behaved like a "classical" Gbg effector, showing very low basal activity and strong Gbg-dependent activation, GIRK1 could not be activated by coexpressed Gbg whilst retaining activation by agonist; Ga_i3GA restored the ability of Gbg to activate GIRK1, indicating that Ga_i (probably as Ga_ibg heterotrimer) allosterically regulates the Gbg gating of GIRK. These results suggest a specific role for GIRK1 as the scaffold for Gabg within GIRK-G protein signaling complex, and imply that the Ga_i/obg heterotrimers are not only precursors of free Gbg, but also active regulators of GIRK gating.