A simple and reliable method is described for the determination of leucine flux in vivo using two stable isotopes of leucine and gas chromatography-mass spectrometry (GC-MS). [6,6,6-2H3]Leucine is administered as a primed-dose constant infusion in vivo and DL-[2H7]-leucine is added to plasma as an internal standard. Plasma leucine concentration and moles per cent enrichment of [2H3]leucine can be determined simultaneously by GC-MS and selected ion monitoring. Leucine flux calculated from the [6,6,6-2H3]leucine data was nearly identical to that obtained with L-[U-14C]leucine in dogs. This method is readily applicable to the study of leucine metabolism in humans of all ages and laboratory animals.