G protein activated K+ channels (GIRK, Kir3) are switched on by direct binding of Gbetagamma following activation of Gi/o proteins via G protein-coupled receptors (GPCRs). Although Galphai 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 Galphai3 decreases the basal activity (Ibasal) and increases the extent of activation by purified or coexpressed Gbegagamma. Here we show that this regulation is exerted by the 'inactive' GDP-bound Galphai3GDP and involves the formation of Galphai3betagamma heterotrimers, by a mechanism distinct from mere sequestration of Gbetagamma 'away' from the channel. The regulation of basal and Gbetagamma-evoked current was produced by the 'constitutively inactive' mutant of Galphai3, Galphai3G203A, which strongly binds Gbetagamma, but not by the 'constitutively active' mutant, Galphai3Q204L, or by Gbetagamma-scavenging proteins. Furthermore, regulation by Galphai3G203A was unique to the GIRK1 subunit; it was not observed in homomeric GIRK2 channels. In vitro protein interaction experiments showed that purified Gbetagamma enhanced the binding of Galphai3GDP to the cytosolic domain of GIRK1, but not GIRK2. Homomeric GIRK2 channels behaved as a 'classical' Gbetagamma effector, showing low Ibasal and strong Gbetagamma-dependent activation. Expression of Galphai3G203A did not affect either Ibasal or Gbetagamma-induced activation. In contrast, homomeric GIRK1* (a pore mutant able to form functional homomeric channels) exhibited large Ibasal and was poorly activated by Gbegagamma. Expression of Galphai3GDP reduced Ibasal and restored the ability of Gbetagamma 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 Galphai3GDP and Gbetagamma, while GIRK2 is a Gbetagamma-effector insensitive to Galphai3GDP.