The cardiac G protein-gated K+ channel, I(KACh), is activated by application of purified and recombinant β and γ subunits (Gβγ) of heterotrimeric G proteins to excised inside-out patches from atrial membranes (Logothetis, D. E., Kurachi, Y., Galper, J., Neer, E., and Clapham, D. E. (1987) Nature 325, 321-326; Wickman, K., Iniguez-Lluhi, J., Davenport, P., Taussig, R. A., Krapivinsky, G. B., Linder, M. E, Gilman, A., and Clapham, D. E. (1994) Nature 368, 255-257). Cardiac I(KACh) is composed of two inward rectifier K+ channel subunits, GIRK1 and CIR (Krapivinsky, G., Gordon, E., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We show that Gβγ directly binds to immunoprecipitated cardiac I(KACh) as well as to recombinant CIR and GIRK1 subunits, with dissociation constants (K(d)) of 55, 50, and 125 nM, respectively. In each case, binding appeared specific as judged by competition of unlabeled Gβγ with radiolabeled Gβγ and inhibition of binding by antigenic peptide or Gα-GDP, but not Gα-GTPγS (guanosine 5'-3-O-(thio)triphosphate). In contrast, Gα (GTPγS- or GDP-bound) did not bind to the native channel. We conclude that Gβγ binds directly and specifically to I(KACh) via interactions with both CIR and GIRK1 subunits to gate the channel.