Although positron-emission tomography (PET) with labeled fatty acid delineates infarct size and permits qualitative assessment of fatty acid utilization, quantification of oxidative metabolism is limited by complex alterations in the pattern of utilization of fatty acid during ischemia and reperfusion. Because metabolism of acetate by myocardium is less complex than that of glucose or palmitate, we characterized kinetics of utilization of radiolabeled acetate in 37 isolated rabbit hearts perfused with modified Krebs-Henseleit buffer and performed a pilot tomographic study in man. Results of initial experiments with carbon-14-labeled acetate (14C-acetate) indicated that the steady-state extraction fraction of acetate averaged 61.5 +/- 4.0% in control hearts (n = 4), 93.6 +/- 0.9% in hearts rendered ischemic (n = 4), and 54.8 +/- 4.0% in hearts reperfused after 60 min of ischemia (n = 3). Oxidation of 14C-acetate, assessed from the rate of efflux of 14CO2 in the venous effluent, correlated closely with the rate of oxygen consumption under diverse metabolic conditions (r = .97, p less than .001). In addition, no significant differences were observed between rates of efflux of total 14C in all chemical species (reflecting total clearance of tracer from myocardium) and efflux of 14CO2. Clearance of 11C-acetate, measured externally with gamma probes in normal and ischemic myocardium, correlated closely with clearance of 14C-acetate measured directly in the effluent (r = .99, p less than .001) and with overall myocardial oxygen consumption (r = .95, p less than .001). Accumulation and clearance of 11C-acetate from human myocardium with PET demonstrated kinetics comparable to those seen with radiolabeled acetate in vitro. Thus externally detectable clearance of 11C-acetate provides a quantitative index of myocardial oxidative metabolism despite variation in the patterns of intermediary metabolism that confounds interpretation of results with conventionally used tracers such as glucose and fatty acid.