These studies were performed 1) to compare two isotopic methods (3H2O and 14CO2 production) of measuring free fatty acid (FFA) oxidation; 2) to determine whether isotopic estimates of fatty acid oxidation during hypoinsulinemia are plausible when compared with those obtained using indirect calorimetry; and 3) to examine whether the delay between the exit of [14C]FFA from plasma and the appearance of 14CO2 in breath is accounted for solely by bicarbonate kinetics. Studies in 11 healthy volunteers revealed that [14C]- and [3H]FFA tracers provide similar estimates of FFA turnover and oxidation. Isotopic estimates of fatty acid oxidation were less than those of indirect calorimetry under basal conditions but equaled or exceeded indirect calorimetry estimates after 3 h of acute hypoinsulinemia (somatostatin induced). After stopping tracer infusions, the half-life of plasma [14C]FFA was 3.7 +/- 0.1 min. The half-life of 14CO2 decay from [14C]bicarbonate was 37 +/- 1 min, and the half-life of 14CO2 decay after discontinuation of [14C]FFA infusion was 141 +/- 10 min. Intracellular preoxidative fatty acid pools (possibly triglycerides) most probably account for the marked delay between the exit of FFA tracers from plasma and the appearance of isotopic markers of oxidation in measurable spaces. This delay can result in erroneous estimates of "FFA oxidation." We conclude that tracer measurements of FFA oxidation are invalid under most circumstances.