ACETYLCHOLINE activates inwardly rectifying potassium channels (I_k.ACh) in the heart¹ through muscarinic receptor binding and activation of pertussis-toxin-sensitive G proteins^2,3. Experiments showing that only the βγ-subunit (Gβγ) activates I_K.ACH (ref. 4) were challenged by reports that only the activated α-subunit (Gα) was effective⁵. Here we examine I_K.ACh regulation using purified brain and recombinant G-protein subunits. Six recombinant Gβγ-subunits activated I_K.ACh with apparent half-maximal activation concentrations of 3-30 nM. Activation of I_K.ACh by recombinant Gα-GTPγS was observed, but this was probably due to release of GTPγS from the protein. Importantly, I_K.ACh activity elicited by GTPγS was inhibited by purified brain and recombinant Gα-GDP, suggesting that native Gβγ plays a major role in this pathway. We conclude that Gβγ is a primary regulator of I_K.ACh activity.