We previously reported that combined blockade of adenosine receptors and ATP-sensitive K+ channels (K+(ATP) channels) blunted but did not abolish the response of coronary blood flow to exercise. This study tested the hypothesis that the residual increase in coronary flow in response to exercise after adenosine receptor and K+(ATP) channel blockade is dependent on endogenous NO. Dogs were studied at rest and during a four-stage treadmill exercise protocol under control conditions, during K+(ATP) channel blockade with glibenclamide (50 μg · kg-1 · min-1 IC) in the presence of adenosine receptor blockade with 8-phenyltheophylline (8-PT, 5 mg/kg IV), and after the addition of the NO synthase inhibitor N(G)-nitro-L-arginine (LNNA, 1.5 mg/kg IC). During control conditions, coronary blood flow was 49±3 mL/min at rest and increased to 92±8 mL/min at peak exercise. LNNA alone or in combination with 8-PT did not alter resting coronary flow and did not impair the normal increase in flow during exercise, indicating that when K+(ATP) channels are intact, neither NO nor adenosine-dependent mechanisms are obligatory for maintaining coronary blood flow. Combined K+(ATP) channel and adenosine blockade decreased resting coronary flow to 27±3 mL/min (P<.05), but exercise still increased flow to 45±5 mL/min (P<.05). The subsequent addition of LNNA further decreased resting coronary flow to 20±2 mL/min and markedly blunted exercise-induced coronary vasodilation (coronary vascular conductance, 0.20±0.03 mL · min-1 · mm Hg-1 at rest versus 0.24±0.04 mL · min-1 · mm Hg-1 during the heaviest level of exercise; P=.22), so that coronary flow both at rest and during exercise was below the control resting level. The findings suggest that K+(ATP) channels are critical for maintaining coronary vasodilation at rest and during exercise but that when K+(ATP) channels are blocked, both adenosine and NO act to increase coronary blood flow during exercise. In the presence of combined K+(ATP) channel blockade and adenosine receptor blockade, NO was able to produce approximately one quarter of the coronary vasodilation that occurred in response to exercise when all vasodilator systems were intact.