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 (MV(O2)) and the ability of endogenous nitric oxide (NO) to modulate MV(O2) during exercise. In chronically instrumented dogs (n = 8), hemodynamic and MV(O2) 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 MV(O2)-triple product (TP) relations and reversed the disproportionate increases in MV(O2) 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. Despite vascular nitrate tolerance, MV(O2)-TP relations were shifted downward compared with pre-NTG exercise. Seven days after NTG withdrawal, vascular responses to boluses of NTG had recovered from tolerance, and MV(O2)-TP relations during exercise were back to pre-NTG level. At that time, blockade of NO formation failed to alter MV(O2)-TP relations. Thus NTG caused a sustained reduction of cardiac MV(O2), independent of metabolic demand during exercise, despite tolerance of the coronary microcirculation. NTG-induced vascular tolerance and MV(O2) reductions were reversible by NTG withdrawal, but endogenous NO-dependent modulation of O2 consumption was severely impaired.