G protein-activated K + channels (Kir3 or GIRK) are activated by direct interaction with Gβγ. Gα is essential for specific signaling and regulates basal activity of GIRK (I basal) and kinetics of the response elicited by activation by G protein-coupled receptors (I evoked). These regulations are believed to occur within a GIRK-Gα-Gβγ signaling complex. Fluorescent energy resonance transfer (FRET) studies showed strong GIRK-Gβγ interactions but yielded controversial results regarding the GIRK-Gα i/o interaction. We investigated the mechanisms of regulation of GIRK byGα i/o using wild-typeGα i3 (Gα i3WT) and Gα i3 labeled at three different positions with fluorescent proteins, CFP or YFP (xFP). Gα i3xFP proteins bound the cytosolic domain of GIRK1 and interacted with Gβγ in a guanine nucleotide-dependent manner. However, only an N-terminally labeled, myristoylated Gα i3xFP (Gα i3NT) closely mimicked all aspects of Gα i3WT regulation except for a weaker regulation of I basal. Gα i3 labeled with YFP within the Gα helical domain preserved regulation of I basal but failed to restore fast I evoked. Titrated expression of Gα i3NT and Gα i3WT confirmed that regulation of I basal and of the kinetics of I evoked of GIRK1/2 are independent functions of Gα i. FRET and direct biochemical measurements indicated much stronger interaction between GIRK1 and Gβγ than between GIRK1 and Gα i3. Thus, Gα i/oβγ heterotrimer may be attached to GIRK primarily via Gβγ within the signaling complex. Our findings support the notion that Gα i/o actively regulates GIRK. Although regulation of I basal is a function of Gα i GDP, our new findings indicate that regulation of kinetics of I evoked is mediated by Gα i GTP.