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Drosophila melanogaster strain Oregon-R* was grown on standard medium supplemented with [U-13 C6]glucose. One to two days after hatching, flies were extracted with water. Glucose was isolated chromatographically from the extract and was analyzed by 13 C NMR spectroscopy. All 13 C signals of the isolated glucose were multiplets arising by 13 C 13 C coupling. Based on a comprehensive analysis of the coupling constants and heavy isotope shifts in glucose, the integrals of individual 13 C signal patterns afforded the concentrations of certain groups of 13 C isotopologs. These data were deconvoluted by a genetic algorithm affording the abundances of all single-labeled and of 15 multiply labeled isotopologs. Among the latter group, seven isotopologs were found at concentrations >0.1 mol % with [1,2-13 C2]glucose as the most prominent species. The multiply 13 Clabeled glucose isotopologs are caused by metabolic remodeling of the proffered glucose via a complex network of catabolic and anabolic processes involving glycolysis and͞or passage through the pentose phosphate, the Cori cycle and͞or the citrate cycle. The perturbation method described can be adapted to a wide variety of experimental systems and isotope-labeled precursors.stable isotope ͉ nuclear magnetic resonance ͉ genetic algorithm ͉ insect ͉ X group