Adenosine (Ado), a smooth muscle vasodilator and modulator of cardiac function, is taken up by many cell types via a saturable transporter, blockable by dipyridamole. To quantitate the influences of endothelial cells in governing the blood-tissue exchange of Ado and its concentration in the interstitial fluid, one must define the permeability-surface area products (PS) for Ado via passive transport through interendothelial gaps [PS_g(Ado)] and across the endothelial cell luminal membrane (PS_ecl) in their normal in vivo setting. With the use of the multiple-indicator dilution (MID) technique in Krebs-Ringer perfused, isolated guinea pig hearts (preserving endothelial myocyte geometry) and by separating Ado metabolites by HPLC, we found permeability-surface area products for an extracellular solute, sucrose, via passive transport through interendothelial gaps [PS_g(Suc)] to be 1.9 ± 0.6 ml · g¹ · min¹ (n = 16 MID curves in 4 hearts) and took PS_g(Ado) to be 1.2 times PS_g(Suc). MID curves were obtained with background nontracer Ado concentrations up to 800 µm, partially saturating the transporter and reducing its effective PS_ecl for Ado. The estimated maximum value for PS_ecl in the absence of background adenosine was 1.1 ± 0.1 ml · g¹ · min¹ [maximum rate of transporter conformational change to move the substrate from one side of the membrane to the other (maximal velocity; V_max) times surface area of 125 ± 11 nmol · g¹ · min¹], and the Michaelis-Menten constant (K_m) was 114 ± 12 µM, where ± indicates 95% confidence limits. Physiologically, only high Ado release with hypoxia or ischemia will partially saturate the transporter.