To determine the pathway of plasma FFA oxidation and the site(s) of label fixation observed during infusion of FFA tracers, [1-13C]palmitate and [1-14C]acetate were infused intravenously for 3 h in five volunteers. Breath 13CO2 enrichment and 14CO2 specific activity were followed for 6 h to determine the labeled CO2 decay rates. Acetate enters directly into the TCA cycle; hence, if palmitate transits a large lipid pool before oxidation, 13CO2 enrichment (from palmitate) should decay slower than 14CO2 specific activity (from acetate). Breath 13CO2 enrichment and 14CO2 specific activity decayed at a similar rate after stopping the tracer infusions (half-lives of 13CO2 and 14CO2 decay: mean [+/- SE] 106.6 +/- 8.9 min, and 96.9 +/- 6.0 min, respectively, P = NS), which suggests that palmitate enters the TCA cycle directly and that label fixation occurs after citrate synthesis. Significant label fixation was shown in plasma glutamate/glutamine and lactate/pyruvate during infusion of either [1,2-13C]acetate or [U-13C]palmitate, suggesting that TCA cycle exchange reactions are at least partly responsible for label fixation. This was consistent with our finding that the half-lives of 13CO2 enrichment and 14CO2 specific activity decreased significantly during exercise to 14.4 +/- 3 min and 16.8 +/- 1 min, respectively, since exercise significantly increases the rate of the TCA cycle in relation to that of the TCA cycle exchange reactions. We conclude that plasma FFA entering cells destined to be oxidized are directly oxidized and that tracer estimates of plasma FFA oxidation will underestimate the true value unless account is taken of the extent of label fixation.