We have previously demonstrated that pertussis toxin (PTX)-sensitive G protein (G_PTX) plays a major role in coronary microvascular vasomotion during hypoperfusion. We aimed to elucidate the role of G_PTX during increasing metabolic demand. In 18 mongrel dogs, coronary arteriolar diameters were measured by fluorescence microangiography using a floating objective. Myocardial oxygen consumption (MO₂) was increased by rapid left atrial pacing. In six dogs, PTX (300 ng/ml) was superfused onto the heart surface for 2 h to locally block G_PTX. In eight dogs, the vehicle (Krebs solution) was superfused in the same way. Before and after each treatment, the diameters were measured during control (130 beats/min) and rapid pacing (260 beats/min) in each group. Metabolic stimulation before and after the vehicle treatment caused 8.6 ± 1.8 and 16.1 ± 3.6% dilation of coronary arterioles <100 µm in diameter (57 ± 8 µm at control, n = 10), respectively. PTX treatment clearly abolished the dilation of arterioles (12.8 ± 2.5% before and 0.9 ± 1.6% after the treatment, P < 0.001 vs. vehicle; 66 ± 8 µm at control, n = 11) in response to metabolic stimulation. The increases in MO₂ and coronary flow velocity were comparable between the vehicle and PTX groups. In four dogs, 8-phenyltheophylline (10 µM, superfusion for 30 min) did not affect the metabolic dilation of arterioles (15.3 ± 2.0% before and 16.4 ± 3.8% after treatment; 84.3 ± 11.0 µm at control, n = 8). Thus we conclude that G_PTX plays a major role in regulating the coronary microvascular tone during active hyperemia, and adenosine does not contribute to metabolic vasodilation via G_PTX activation.