The purpose of this study was to determine the changes in cardiac interstitial fluid (ISF) purine metabolites during 90 min of regional myocardial ischemia. To collect ISF metabolites and measure local coronary blood flow (CBF), cardiac microdialysis probes were implanted into the left anterior descending artery (LAD) and left circumflex artery (LC) perfused myocardium of chloraloseurethane anesthetized dogs (n = 7). Regional ventricular wall thickness was measured in the LAD and LC perfused regions with sonomicrometric crystals, using systolic wall thickening (SWT) as an index of regional ventricular function. Regional myocardial ischemia, produced by occlusion of the LAD, resulted in a decrease in CBF (hydrogen clearance) from 77.3 ± 12.4 to 10.9 ± 4.4 ml/min/100g (P < 0.05), and systolic wall thinning (control SWT = 15.5 ± 2.2%; ischemic SWT = −6.8 ± 1.7%). ISF adenosine was transiently elevated in the ischemic region, obtaining a maximum sixfold increase after 15 min of ischemia. Inosine, hypoxanthine, and to a lesser extent xanthine, composed the majority of metabolites which accumulated in the ISF of the ischemic region, accounting for greater than 95% of the total purine metabolites in the ISF after 20 min of ischemia. Despite the marked increase in ISF inosine, hypoxanthine, and xanthine levels, ISF uric acid levels did not increase in the ischemic region. Although CBF and SWT did not change in the nonischemic LC perfused area, there were small transient increases (two- to fourfold) in ISF adenosine, inosine, and hypoxanthine levels. In summary, these data demonstrate that purine metabolites accumulate rapidly in the ISF during myocardial ischemia. Cardiac microdialysis provides a sensitive, selective, and simple means to assess metabolic alterations associated with ischemia.