In this study we determined the rate of conversion of carbon-labeled acetate to carbon dioxide in normal human volunteers and in anesthetized dogs. In human subjects (n = 4), [1-13C]acetate was infused on one occasion, and [2-13C]acetate was infused in the repeat study in the same subjects. In postabsorptive volunteers (n = 6), 81.2 +/- 6.5% (mean +/- SEM) and 53.1 +/- 7.4% of infused 13C was recovered as 13CO2 when [1-13C]- or [2-13C]acetate, respectively, were infused. In one subject studied during exogenous glucose infusion at 3.5 mg.kg-1.min-1, recovery of 13CO2 was 72.7% and 38.5% from [1-13C]- and [2-13C]acetate, respectively. In dogs, [1-14C]- and [2-13C]acetate were infused simultaneously. Recovery of 14CO2 was 75.9 +/- 2.5% of infused isotope whereas recovery of 13CO2 was 40.8 +/- 1.9%. We concluded that the position of the label in acetyl coenzyme A (CoA) determines the extent to which the process of oxidation of labeled acetyl CoA is reflected in labeled carbon dioxide excretion.