Characterization of adenosine's role as a regulator of coronary blood flow requires accurate measurement of endogenous adenosine concentration in the left ventricular (LV) interstitial compartment. Existing techniques for determining adenosine in this compartment are indirect, requiring the acceptance of major assumptions before conclusions can be drawn. We describe a new technique utilizing a LV epicardial diffusion well that allows us to make rapid, direct measurement of LV interstitial adenosine concentration, avoiding many problems inherent in existing techniques. Our results show adenosine concentrations of 555 pmol/ml in resting anesthetized dogs, indicating a resting adenosine level well within the vasoactive range. Further experiments using intramyocardial bolus injections of methylene blue dye and [8-14C]adenosine indicate that the epicardial well receives adenosine from a transmural distribution of LV interstitium and not from epicardial sources only. The transmural interstitial adenosine is transported via small lymphatics to the epicardial surface of the heart where diffusion occurs into the epicardial well. We also examined diffusion characteristics of the parietal pericardial membrane and found that the rate constant of adenosine diffusion for this and the visceral pericardium are of the same order of magnitude, indicating that the extensively used standard pericardial superperfusate method probably underestimates cardiac interstitial adenosine concentration by 50% or more. The influence of the parietal pericardium adequately explains why our resting adenosine concentrations using the epicardial well are higher than those recently reported using the standard pericardial superperfusate method.