We have previously shown that direct binding of the betagamma subunit of G protein (G betagamma) to both the N-terminal domain and the C-terminal domain of a cloned G protein-gated inward-rectifying K+ channel subunit, GIRK1, is important for channel activation. We have now further localized the G betagamma binding region in the N-terminal domain of GIRK1 to amino acids 34-86 and the G betagamma binding region in the C-terminal domain of GIRK1 to two separate fragments of amino acids 318-374 and amino acids 390-462. Of the four cloned mammalian GIRK subunits, GIRK1-4, GIRK1 and 4 form heteromeric K+ channels in the heart and similar channels in the brain include heteromultimers of GIRK1 and 2, and possibly other GIRK homomultimers and heteromultimers. We found that the N-terminal and the C-terminal domains of all four GIRKs bound G betagamma. The G betagamma binding activities for the C-terminal domains of GIRK2-4 were lower than that for the C-terminal domain of GIRK1. The higher G betagamma binding activity for the C-terminal domain of GIRK1 is due to amino acids 390-462 which are unique to GIRK1. We also found that the N-terminal and C-terminal domains of GIRKs interacted with each other, and the N-terminal domain of either GIRK1 or GIRK4 together with the C-terminal domain of GIRK1 exhibited much enhanced binding of G betagamma. These results are consistent with the idea that the N- and C-terminal domains of the cardiac G protein-gated K+ channel subunits may interact with each other to form higher affinity binding site(s) for G betagamma.