The objectives of the present study were first, to determine the coronary microvascular response to vagal stimulation and to compare it to exogenously administered acetylcholine, and second, to determine the microvascular response to larger doses of acetylcholine which preferentially increase subendocardial blood flow. In anesthetized cats and dogs, the left ventricular epicardial vasculature was visualized in mid-diastole with stroboscopic epi-illumination. Myocardial perfusion was measured with the radioactive microsphere technique. In cats microvascular diameters were measured at control and following bilateral vagal nerve stimulation (5-30 Hz) or left atrial infusion of acetylcholine (0.4-1.0 micrograms/kg/min). Aortic pressure and heart rate (A-V-sequential pacing) were maintained constant. Vagal stimulation (n = 13) increased myocardial perfusion by 25 +/- 9% (control: 161 +/- 17 ml/min x 100 g). Acetylcholine (n = 13) produced a similar increase in myocardial flow (control: 185 +/- 16 ml/min x 100 g, 30 +/- 9%). Both vagal stimulation and acetylcholine dilated all size arteries and arterioles (51-410 microns; 5 +/- 1% and 11 +/- 2%, respectively). In dogs intracoronary administration of acetylcholine (10 micrograms/min) that increased myocardial flow twofold (control: 129 +/- 7 ml/min x 100 g; 10 micrograms/min: 263 +/- 26 ml/min x 100 g) and increased the endo/epi flow ratio also dilated all vessel sizes. In conclusion, vagal stimulation and exogenously administered acetylcholine produce similar effects on the coronary microcirculation and dilate all size classes of arteries and arterioles in a similar manner. Intracoronary infusion of acetylcholine which preferentially increases subendocardial blood flow also dilates all size classes of microvessels. Thus, the ability of acetylcholine to preferentially increase subendocardial blood flow cannot be explained by a selective dilation of a particular size class of arterioles. These data suggest that neurally released acetylcholine can diffuse to the endothelial layer to release vasodilator substances in vivo.