A regulating mechanism of the ATP-sensitive potassium channels (KATP channels) is yet to fully explained. This study was carried out to investigate the effects of intracellular application of monocarboxylates (acetate, formate, lactate, and pyruvate) on KATP channels in isolated rabbit ventricular myocytes. Single channel currents of KATP channels were recorded using the excised inside-out or permeabilized attached (open-cell) patch-clamp technique at room temperature. Intracellular application of acetate, formate and pyruvate led to an inhibition of channel activity, whereas intracellular application of lactate increased channel activity. These effects were reversible upon washout. Analysis of single channel kinetics showed that monocarboxylates did not affect open-time constant and close-time constant. These results suggest that monocarboxylates participate in modulating KATP channels activity in cardiac cells and that modulation of KATP channels activity may resolve the discrepancy between the low Ki in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.