We describe a simple method using 13 C labeling and NMR spectroscopy to determine the flux contributions of alternative pathways in Saccharomyces cerevisiae that produce the same metabolite with identical labeling patterns. Cells were incubated with a 13 C-labeled precursor for one of the branches, and the absolute enrichment of the product and its metabolic precursor(s) was quantified. The ratio of the absolute enrichment of the product to that of its precursor reflects the contribution of the pathway. The method was applied to the biosynthesis of glycine in yeast, which can occur from threonine via threonine aldolase or from serine via serine hydroxymethyltransferase. [2-13 C]Aspartate and [2-13 C]serine were used as labeled precursors for the threonine aldolase and serine hydroxymethyltransferase pathways, respectively. The data show that in cells possessing both pathways, the serine hydroxymethyltransferase pathway contributes 65-75% of the total glycine production. In comparison with other approaches, this method provides an inexpensive, flexible alternative to determining the flux contributions of split pathways under controlled conditions and should have wide applicability in the metabolic engineering of microorganisms.