The response of the steady-state level of mitochondrial NAD(P)H of individual cardiac myocytes to substrate and to pharmacological alteration of intracellular calcium was investigated using a defined pacing protocol. Rapid pacing (5 Hz) reversibly decreased the NAD(P)H level and increased oxygen consumption whereas phosphocreatine and ATP levels did not change significantly. Verapamil plus NiCl2 blockade of calcium channels abolished contractions. Ryanodine, which prevents calcium-induced calcium release, also stopped cell contraction. NAD(P)H levels do not change in the absence of contraction. Blockade of sarcolemmal K+ channels did not stop contraction, and NAD(P)H levels reversibly decreased during rapid pacing. Thus rapid contractions are associated with a reversible decrease in NAD(P)H levels. Ruthenium red blockade of Ca2+ entry into mitochondria did not block contraction but significantly decreased NAD(P)H levels in both slowly paced (0.5 Hz) and rapidly paced cells. The simplest explanation of these data is that the steady-state reduction of NAD(P)H is strongly dependent on the rate of ATP utilization and not on sarcoplasmic Ca2+ levels when the oxygen and substrate supplies are not limiting and the intracellular calcium regulation is maintained. An effect of intracellular Ca2+ on NAD(P)H is observed only when Ca2+ entry into mitochondria is blocked with ruthenium red.