Acetylcholine released during parasympathetic stimulation of the vagal nerve slows the heart rate through the activation of muscarinic receptors and subsequent opening of an inwardly rectifying potassium channel. The activation of these muscarinic potassium channels is mediated by a pertussis toxin-sensitive heterotrimeric GTP-binding protein (G protein). It has not been resolved whether exogenously applied G alpha or G beta gamma, or both, activate the channel. Using a heterologous expression system, we have tested the ability of different G protein subunits to activate the cloned muscarinic potassium channel, GIRK1. We report here that coexpression of GIRK1 with G beta gamma but not G alpha beta gamma in Xenopus oocytes results in channel activity that persists in the absence of cytoplasmic GTP. This activity is reduced by fusion proteins of the beta-adrenergic receptor kinase and of recombinant G alpha i-GDP, both of which are known to interact with G beta gamma. Moreover, application of recombinant G beta gamma, but not G alpha i-GTP-gamma S, activates GIRK1 channels. Thus G beta gamma appears to be sufficient for the activation of GIRK1 muscarinic potassium channels.