Cholinergic muscarinic, serotonergic, opioid and several other G-protein-coupled neurotransmitter receptors activate inwardly rectifying K+ channels of the GIRK family, slowing the heartbeat and decreasing the excitability of neuronal cells’. Inhibitory modulation of GIRKs by G-protein-coupled receptors may have important implications in cardiac and brain physiology, Previously G alpha and G beta gamma subunits of heterotrimeric G proteins have both been implicated in channel opening(2,3), but recent studies attribute this role primarily to the G beta gamma dimer that activates GIRKs in a membrane-delimited fashion, probably by direct binding to the channel protein(4-8). We report here that free GTP gamma S-activated G(alpha i1), but not G(alpha i2) or G(alpha i3), potently inhibits G(beta 1 gamma 2)-induced GIRK activity in excised membrane patches of Xenopus oocytes expressing GIRK1. High-affinity but partial inhibition is produced by G(alpha s)-GTP gamma S. G(alpha i1)-GTP gamma S also inhibits G(beta 1 gamma 2)-activated GIRK in atrial myocytes. Antagonistic interactions between G(alpha) and G(beta gamma) may be among the mechanisms determining specificity of G protein coupling to GIRKs.