The aim of this study was to investigate the effect of NADH-supplementation on the metabolic condition of isolated guinea pig ventricular cardiomyocytes using the pinacidil-primed IK(ATp) as an indicator of subsarcolemmal ATP concentration. Membrane currents were studied using the patch-clamp technique in the whole-cell recording mode at 36-37oC. Under physiological conditions (4.3 mM ATP in the pipette solution, ATPj) IK(ATP) did not contribute to basal electrical activity The K(ATP) channel opener pinacidil activated IK(ATP) dependent on [ATP]i showing a significantly more pronounced activation at lower (1 mM) [ATP]j. Incubation of cardiomyocytes with 300 μg/mI NADH (4-6 h) resulted in a significantly reduced IK(ATp) activation by pinacidil compared to control cells. Equimolar amounts of the related compounds nicotinamide and NAD+ were not able to achieve a similar effect like NADH. These data show that incubation of guinea pig ventricular cardiomyocytes with NADH results in a decreased activation Of IK(ATp) by pinacidil compared to control myocytes indicating a higher subsarcolemmal ATP concentration due to NADH -supplementation. Measurement of adenine nucleotides by HPLC revealed a significant increase in intracellular ATP (NADH supplementation: 45.59 ± 1,88 nmol/mg protein versus control: 35.35 ± 2.57 nmol/mg protein, P < 0.000005).