Adenosine is a key myocardial metabolite that elicits coronary vasodilation in a variety of pathophysiological conditions. We examined the mechanism of adenosine-induced vasodilation in coronary arterioles from patients with heart disease. Human coronary arterioles (HCAs) were dissected from pieces of the atrial appendage obtained at the time of cardiac surgery and cannulated for the measurement of internal diameter with videomicroscopy. Adenosine-induced vasodilation was not inhibited by endothelial denudation, but A(2) receptor antagonism with 3,7-dimethyl-1-propargylxanthine and adenylate cyclase (AC) inhibition with SQ22536 significantly attenuated the dilation. In contrast, A(1) receptor antagonism with 8-cyclopentyl-1,3-dipropylxanthine significantly augmented the sensitivity to adenosine. Moreover, dilation to A(2a) receptor activation with 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine hydrochloride was reduced by the A(1) receptor agonist (2S)-N(6)-(2-endo-norbornyl)adenosine. The nonspecific calcium-activated potassium (K(Ca)) channel blocker tetrabutylammonium attenuated adenosine-induced dilation, as did the intermediate-conductance K(Ca) blocker clotrimazole. Neither the large-conductance K(Ca) blocker iberiotoxin nor small-conductance K(Ca) blocker apamin altered the dilation. In conclusion, adenosine endothelium independently dilates HCAs from patients with heart disease through a receptor-mediated mechanism that involves the activation of intermediate-conductance K(Ca) channels via an AC signaling pathway. The roles of A(1) and A(2) receptor subtypes are opposing, with the former being inhibitory to AC-mediated dilator actions of the latter. These observations identify unique fundamental physiological characteristics of the human coronary circulation and may help to target the use of novel adenosine analogs for vasodilation in perfusion imaging or suggest new strategies for myocardial preconditioning.