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 (MVO(2)) 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 microm in diameter (57 +/- 8 microm 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 microm at control, n = 11) in response to metabolic stimulation. The increases in MVO(2) and coronary flow velocity were comparable between the vehicle and PTX groups. In four dogs, 8-phenyltheophylline (10 microM, 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 microm 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.