The long-term benefits of nitroglycerin (NTG) therapy are limited by the development of vascular tolerance and endothelial dysfunction in conductance coronary arteries. We have determined whether nitrate tolerance extends to NTG effects on myocardial O₂ consumption (MVo₂) and the ability of endogenous nitric oxide (NO) to modulate MVo₂ during exercise. In chronically instrumented dogs (n=8), hemodynamic and MVo₂ responses to treadmill exercise were measured before, during tolerance (3 and 7 days of NTG delivery) and 7 days after NTG withdrawal. Acute NTG delivery caused a parallel downward shift of the MVo₂/triple product (TP) relations and reversed the disproportionate increases in MVo₂ caused by the blockade of NO formation. After 7 days of continuous transdermal NTG delivery, vascular tolerance was displayed as a >75% reduction of coronary blood flow (CBF) responses to NTG boluses. In spite of vascular nitrate tolerance, MVo₂/TP relations were shifted downward when compared to pre-NTG exercise. Seven days after NTG withdrawal, vascular responses to boluses of NTG had recovered from tolerance and MVo₂/TP relations during exercise were back to pre-NTG level. At that time, blockade of NO formation failed to alter MVo₂/TP relations. Thus, NTG caused a sustained reduction of cardiac MVo₂, independent of metabolic demand, during exercise, in spite of tolerance of the coronary microcirculation. NTG-induced vascular tolerance and MVo₂ reductions were reversible by NTG withdrawal but endogenous NO-dependent modulation of O₂ consumption was severely impaired.