G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gβγ following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although Gα i subunits do not activate GIRKs, they interact with the channels and regulate the gating pattern of the neuronal heterotetrameric GIRK1/2 channel (composed of GIRK1 and GIRK2 subunits) expressed in Xenopus oocytes. Coexpressed Gαi3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gβγ. Here we show that this regulation is exerted by the 'inactive' GDP-bound Gαi3GDP and involves the formation of Gαi3βγ heterotrimers, by a mechanism distinct from mere sequestration of Gβγ 'away' from the channel. The regulation of basal and Gβγ-evoked current was produced by the 'constitutively inactive'mutant of Gαi3, Gαi3 G203A, which strongly binds Gβγ, but not by the 'constitutively active' mutant, Gαi3 Q204L, or by Gβγ-scavenging proteins. Furthermore, regulation by Gαi3 G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gβγ enhanced the binding of Gαi3 GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gβγ effector, showing low I basal and strong Gβγ-dependent activation. Expression of Gαi3G203A did not affect either Ibasal or Gβγ-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gβγ. Expression of Gαi3 GDP reduced Ibasal and restored the ability of Gβγ to activate GIRK1*, like in GIRK1/2. Transferring the unique distal segment of the C terminus of GIRK1 to GIRK2 rendered the latter functionally similar to GIRK1*. These results demonstrate that GIRK1 containing channels are regulated by both Gαi3 GDP and Gβγ, while GIRK2 is a Gβγ-effector insensitive to Gαi3 GDP.