It has generally been assumed, from assessment of myocardial metabolism with [1-11C]palmitate and positron emission tomography, that clearance of the radiolabel from the myocardium is attributable solely to efflux of the products of oxidative metabolism. However, interpretations would differ if this assumption were unfulfilled. Furthermore, efflux of metabolized and nonmetabolized tracer has not been quantified. Accordingly, in this study, myocardium was perfused extracorporeally in 21 open-chest anesthetized dogs, and the extraction and clearance of [1-11C]palmitate were characterized under baseline conditions (normoxia, n = 21), and, again, with ischemia (n = 6), with hypoxia (n = 9), or under control conditions (n = 6). After intracoronary bolus injection of [1-11C]palmitate, myocardial time activity curves were measured with a β-probe, and the products of oxidative metabolism (11CO2) and efflux of extracted but nonmetabolized fatty acid ('back-diffusion' of [1-11C]palmitate) were measured directly from analysis of arterial and regional coronary venous blood. Under control conditions, 45.2 ± 3.8% (mean ± SD) of initially extracted [1-11C]palmitate was metabolized to 11CO2, whereas 6.2 ± 2.6% back-diffused in unaltered form 1-10 minutes. In contrast, with ischemia (perfusion of 26% of baseline), only 16.9 ± 9.8% of administered tracer evolved as 11CO2 (P < 0.001 compared with control) but 15.6 ± 8.9% (i.e., almost half of the total amount cleared) evolved unaltered as [1-11C]palmitate (P < 0.05). Similarly, with hypoxia, 15.1 ± 8.4% evoled as 11CO2 (P < 0.0001) and 18.8 ± 11.7% back-diffused (P < 0.001). Overall, from 1-40 minutes after intracoronary injection of tracer, back-diffusion of [1-11C]palmitate contributed 40.6% of total radioactivity in the effluent with ischemia, 48.7% with hypoxia, but only 8.9% under control conditions. Despite the increase back-diffusion of [1-11C]palmitate seen with ischemia and hypoxia, the overall residue of 11C activity in myocardium increased, consistent with the diminished clearance observed in the myocardial time-activity curves and the increase in the tissue content of triglyceride and nonesterified fatty acid. Our results indicate that estimates of oxidative metabolism based upon clearance of radiolabeled fatty acid must take into account the efflux of initially extracted but nonmetabolized fatty acid. The findings apply to external determination of oxidative metabolism of the heart with any imaging modality that delineates retention and clearance of labeled fatty acids or their analogs.