While opening of cardiac muscarinic K+ channels is mediated by a guanine nucleotide-binding protein, GK, and normally requires both ACh and cytosolic GTP, we demonstrate that in the absence of agonist and G-nucleotides guinea-pig atrial muscarinic K+ channels are activated by cytosolic ATP (K50 = 224 microM). This activation involves a phosphorylating reaction and is most probably related to a transphosphorylation by a nucleoside diphosphate kinase (NDPK) from ATP to GK-bound GDP. This ATP-induced response is completely inhibited by nanomolar concentrations of GDP or GTP, suggesting a G-nucleotide specific binding site on NDPK.