Acetylcholine signaling through muscarinic type 2 receptors activates atrial G protein-gated inwardly rectifying K ؉ (Kir3) channels via the ␥ subunits of G proteins (G␥). Different combinations of recombinant G␥ subunits have been shown to activate Kir3 channels in a similar manner. In native systems, however, only G␥ subunits associated with the pertussis toxinsensitive G␣ i/o subunits signal to K ؉ channels. Additionally, in vitro binding experiments supported the notion that the C terminus of Kir3 channels interacts preferentially with G␣ i over G␣ q . In this study we confirmed in two heterologous expression systems a preference of G␣ i over G␣ q in the activation of K ؉ currents. To identify determinants of G␥ signaling specificity, we first exchanged domains of G␣ i and G␣ q subunits responsible for receptor coupling selectivity and swapped their receptor coupling partners. Our results established that the G proteins, regardless of the receptor type to which they coupled, conferred specificity to Kir3 activation. We next tested signaling through chimeras between the G␣ i and G␣ q subunits in which the N terminus, the helical, or the GTPase domains of the G␣ subunits were exchanged. Our results revealed that the helical domain of G␣ i (residues 63-175) in the background of G␣ q could support Kir3 activation, whereas the reverse chimera could not. Moreover, the helical domain of the G␣ i subunit conferred "G␣ i -like" binding of the Kir3 C terminus to the G␣ q subunits that contained it. These results implicate the helical domain of G␣ i proteins as a critical determinant of G␥ signaling specificity.