The molecular basis of G-protein inhibition of inward rectifier K + currents was examined by co-expression of G-proteins and cloned Kir2 channel subunits in Xenopus oocytes. Channels encoded by Kir2.3 (HRK1/HIR/BIRK2/BIR11) were completely suppressed by co-expression with G- protein βγ subunits, whereas channels encoded by Kir2.1 (IRK1), which shares 80% amino acid identity with Kir2.3, were unaffected. Co-expression of Gα(i1) and Gα(q) subunits also partially suppressed Kir2.3 currents, but Gα(t), Gα(s), and a constitutively active mutant of Gα(i1) (Q204L) were ineffective. Gβγ and Kir2.3 subunits were co-immunoprecipitated using an anti-Kir2.3 antibody. Direct binding of G-protein βγ subunits to fusion proteins containing Kir2.3 N terminus, but not to fusion proteins containing Kir2.1 N terminus, was also demonstrated. The results are consistent with suppression of Kir2.3 currents resulting from a direct protein-protein interaction between the channel and G-protein βγ subunits. When Kir2.1 and Kir2.3 subunits were coexpressed, the G-protein inhibitory phenotype of Kir2.3 was dominant, suggesting that co-expression of Kir2.3 with other Kir subunits might give rise to novel G-protein-inhibitable inward rectifier currents.