Objectives: In vivo studies of the human coronary resistance circulation cannot control for indirect effects of myocardial metabolism, compression, and neurohumoral influences. This study directly examined the vasodilator responses of the human coronary microcirculation to both receptor-dependent and -independent agonists. Methods: Atrial arterioles were dissected from human right atrial appendage (103±2 μm diameter, n=185 vessels from 145 patients) obtained at the time of cardiopulmonary bypass and left ventricular vessels from explanted human hearts (148±10 μm diameter, n=57 vessels from 18 patients). After dissection, vessels were mounted onto pipettes in Kreb's buffer under conditions of zero flow and at a constant distending pressure of 60 mmHg. Drugs were applied extraluminally and steady state changes in diameter measured with videomicroscopy. Results: After contraction by endothelin or spontaneous tone, increasing concentrations of adenosine diphosphate (ADP) produced a similar dose-dependent dilation in vessels from atria (maximum 89±4%, n=76) and ventricles (maximum 74±9%, n=10). The dilation to ADP was abolished by mechanical removal of the endothelium. Similar dilator responses were found to bradykinin, substance P, arachidonic acid, and the calcium ionophore A23187 in both atria and ventricle. In contrast, acetylcholine (ACh) constricted all atrial vessels (−58±3%, n=63) regardless of patient age or underlying disease. This constriction was attenuated by denudation, but not affected by inhibition of nitric oxide synthase or cyclo-oxygenase. Microvessels isolated from human ventricle exhibited a heterogeneous response to ACh with dilation being the predominant response. Conclusions: We conclude that isolated human coronary arterioles demonstrate endothelium-dependent dilation. However, the response to acetylcholine is unique with vasoconstriction in atrial vessels and dilation in ventricular arterioles.