The membrane background conductance produced by spontaneous opening of the muscarinic K-+ channels was investigated by recording whole-cell and single-channel currents using isolated sino-atrial node cells of rabbits. Increasing the external [K+] from 5.4 mM to 145 mM induced a large inward shift of the whole-cell current accompanied by considerable current fluctuations at -50 m V. Stationary and nonstationary fluctuation analysis indicated that the high-K+-induced current was produced by the spontaneous opening of the muscarinic K+ channel which is mediated by a membrane-bound nucleoside diphosphate kinase. The background conductance due to the muscarinic K+ channel was estimated from the non-stationary fluctuation analysis in the 5.4 mM K+ solution. We concluded that the spontaneous opening of the muscarinic K+ channel is attributable to the background K+ conductance and affects the slow diastolic depolarization in sino-atrial node cells.