Guinea pig or rabbit atrial muscarinic K+ channels in cell-free inside-out patches can be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg2+. This ATP-dependent activation is compatible with the existence of a membrane-bound nucleoside diphosphate kinase (NDPK), which directly phosphorylates GK-bound GDP. We show that this ATP-dependent activation is also possible in frog atrial cells, and that atrial membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphosphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP-dependent activation of muscarinic K+ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction.