Background: Beta-adrenergic stimulation increases Ca2+ transients in cardiomyocytes, demanding elevated ATP generation. This is matched by mitochondrial Ca2+ uptake stimulating Krebs cycle dehydrogenases to regenerate NADH and FADH2, the electron donors for the respiratory chain. NO competes with O2 to slow down electron transfer at the respiratory chain. NO is produced by NO synthases (NOS), with nNOS and eNOS dominating in cardiomyocytes, while the existence of a mitochondrial NOS (mtNOS) is controversial. Since nNOS, eNOS and putative mtNOS are regulated by Ca2+, we speculated that endogenous NO controls respiration during beta-adrenergic stimulation.Methods and Results: Experiments were performed on murine and guinea-pig mitochondria or cardiomyocytes. In mitochondria, ADP accelerated O2 consumption and oxidized NADH, while the NO-donor spermine-NONOate inhibited ADP-induced respiration and reduced NADH. Cardiomyocytes were loaded with NO-sensitive DAF-DA, which locates to cytosol and mitochondria, and paced at 0.5 Hz. Isoproterenol plus elevation of stimulation frequency to 5 Hz increased cellular DAF-DA fluorescence by ∼12% within 3 minutes, which was abrogated by inhibition or genetic ablation of nNOS, but not eNOS. Dialyzing myocytes with DAF-free pipette solution eliminated >50% of DAF fluorescence, with remaining DAF signals deriving from mitochondria. After beta-adrenergic stimulation, a smaller increase in DAF fluorescence remained, which was abrogated by nNOS KO, but not by Ru360 (1µM in pipette), a blocker of the mitochondrial Ca2+ uniporter. The redox states of NADH and FADH2 were dynamically regulated after isoproterenol/5Hz, but not different after nNOS inhibition or KO.Conclusions: During beta-adrenergic stimulation, most endogenous NO derives from nNOS, but not eNOS, while mtNOS plays no role under these conditions. Although NO inhibits mitochondrial respiration in isolated mitochondria, the endogenous concentrations produced in myocytes during beta-adrenergic stimulation do not affect respiration.