Low intravenous infusion rates of [U-13C]palmitate were used to measure systemic palmitate flux at rest (0.5 nmol. kg-1. min-1) and during exercise (2 nmol. kg-1. min-1) in healthy adults. The 13C enrichment of plasma palmitate was determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Palmitate flux was also measured using palmitate radiotracers, which were infused simultaneously to serve as a reference method. Flux values obtained using the two different methods were virtually identical. Overnight postabsorptive palmitate flux was measured on four occasions in three individuals; the flux values were highly reproducible with a coefficient of variation of 11% and 12% for intra-day measurement, and 13% and 8% for inter-day measurement, for [1-(14)C]palmitate and [U-(13)C]palmitate, respectively. During exercise, palmitate turnover measured with [U-(13)C]palmitate and [3H]palmitate were well correlated (r = 0.91). Compared to conventional stable isotope approaches, (e.g., [1-(13)C]palmitate analyzed by GC/MS), [U-(13)C]palmitate reduces the tracer dose by two orders of magnitude when the samples are analyzed with GC/C/IRMS. Uniformly labeled [13C]palmitate is suitable for the measurement of free fatty acid flux in humans.