The determination of substrate turnover rate with stable isotope-labeled compounds has advantages of being safe and applicable in the study of children and pregnant women. Currently, a majority of these studies has been performed with primed constant-rate infusion technique. The isotope enrichment of the substrate in the plasma is measured by gas chromatography-mass spectrometry (GCMS). The turnover rate is then calculated from steady-state kinetics. A number of different equations have been used by various investigators for this purpose. Based on theoretical consideration and experimental data, it is concluded that the equation P = [(1/E) - 1] I or P = (y/x) I should be used for a correct turnover rate calculation, where P is turnover rate in mumol X kg-1, E the isotope enrichment, I the infusion rate in mumol X kg-1 X min-1, and y/x the mole ratio of tracee to tracers. A GCMS standard curve constructed from isotope enrichment versus isotope peak abundance should be used for the former equation, whereas a standard curve constructed from mole ratio (x/y) versus isotope ratio should be used for the latter equation. Interchange of standard curves or use of other equations will produce erroneous turnover rate. This is especially significant when a low enriched isotope-labeled compound is used as a tracer.