G protein-activated K+ channels (GIRK) mediate postsynaptic inhibitory effects of neurotransmitters in the atrium and in the brain by coupling to G protein-coupled receptors (GPCRs). In neurotransmitter-dependent GIRK signalling, Gβγ is released from the heterotrimeric Gαβγ complex upon GPCR activation, activating the channel and attenuating its rectification. Now it becomes clear that Gα is more than a mere Gβγ donor. We have proposed that Gαi3-GDP regulates GIRK gating, keeping its basal activity low but priming (predisposing) the channel for activation by agonist in intact cells, and by Gβγ in excised patches. Here we have further investigated GIRK priming by Gαi3 using a model in which the channel was activated by coexpression of Gβγ, and the currents were measured in intact Xenopus oocytes using the two-electrode voltage clamp technique. This method enables the bypass of GPCR activation during examination of the regulation of the channel in intact cells. Using this method, we further characterize the priming phenomenon. We tested and excluded the possibility that our estimates of priming are affected by artifacts caused by series resistance or large K+ fluxes. We demonstrate that both Gαi3 and membrane-attached Gβγ scavenger protein, m-phosducin, reduce the basal channel activity. However, Gαi3 allows robust channel activation by coexpressed Gβγ, in sharp contrast to m-phosducin, which causes a substantial reduction in the total Gβγ-induced current. Furthermore, Gαi3 also does not impair the Gβγ-dependent attenuation of the channel rectification, in contrast to m-phosducin, which prevents this Gβγ-induced modulation. The Gαi3-induced enhancement of direct activation of GIRK by Gβγ, demonstrated here for the first time in intact cells, strongly supports the hypothesis that Gαi regulates GIRK gating under physiological conditions.