We tested whether hypoxia-induced coronary artery dilation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F₂ 10^-5 M, were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analyzed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyzer. The exchange fraction of [¹⁴C]adenosine over the microdialysis membrane increased from 0.32±0.02 to 0.46±0.02 (n=4, P<0.01) during the study period. At baseline, interstitial adenosine was in the range of 10 nM which is significantly less than previously found myocardial concentrations. Hypoxia (PO₂ 30 mm Hg for 60 min, n=5) increased coronary diameters by 20.0±2.6% (vs. continuous oxygenation -3.1±2.4%, n=6, P<0.001) but interstitial adenosine concentration fell. Blockade of adenosine deaminase (with EHNA, 5 µM), adenosine kinase (with ITU, 10 µM) and adenosine transport (with NBTI, 1 µM) increased interstitial adenosine but the increase was unrelated to hypoxia or diameter. A coronary dilation similar to that during hypoxia could be obtained with 30 µM of adenosine in the organ bath and the resulting interstitial adenosine concentrations (n=5) were 20 times higher than the adenosine concentration measured during hypoxia. Adenosine concentration-response experiments showed vasodilation to be more pronounced during hypoxia (n=9) than during normoxia (n=9, P<0.001) and the A_2A receptor antagonist, ZM241385, 20 nM (n=5) attenuated hypoxia-induced vasodilation while the selective A_2B receptor antagonist MRS1754, 20 nM (n=4) had no effect. The lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at A_2A receptor level.