Nitric oxide (NO) and O₂ compete for cytochrome c oxidase at cytochrome oxidase, thus potentially allowing NO to modulate myocardial respiration. We previously observed a decrease of myocardial phosphocreatine (PCr)/ATP during high cardiac workstates, corresponding to an increase in cytosolic free ADP. This study tested the hypothesis that NO inhibition of respiration contributes to this increase of ADP. Infusion of dobutamine + dopamine (DbDp, each 20 µg/kg/min iv) to more than double myocardial oxygen consumption (MVO₂) in open chest dogs caused a decrease of myocardial PCr/ATP measured with ³¹P NMR from 2.04±0.09 to 1.85±0.08 (p<0.05). Inhibition of NO synthesis with N-nitro-L-arginine (LNNA) while the catecholamine infusion continued, caused PCr/ATP to increase to the control value. In a second group of animals, LNNA administered prior to catecholamine stimulation (reverse intervention of the first group) increased PCr/ATP during basal conditions. In these animals LNNA did not prevent a decrease of PCr/ATP at the high cardiac workstate but, relative to MVO₂, PCr/ATP was significantly higher after LNNA. In a third group of animals, pharmacologic coronary vasodilation with carbochromen was used to prevent changes in coronary flow that might alter endothelial NO production. In these animals LNNA again restored the depressed myocardial PCr/ATP ratio during catecholamine infusion. The finding that inhibition of NO production increased PCr/ATP suggests that during high workstates NO inhibition of mitochondrial respiration requires ADP to increase to drive oxidative phosphorylation.