Metabolic engineering of plants has great potential for the low cost production of chemical feedstocks and novel compounds, but to take full advantage of this potential a better understanding of plant central carbon metabolism is needed. Flux studies define the cellular phenotype of living systems and can facilitate rational metabolic engineering. However the measurements usually made in these analyses are often not sufficient to reliably determine many fluxes that are distributed between different subcellular compartments of eukaryotic cells. We have begun to address this shortcoming by increasing the number and quality of measurements that provide (13)C labeling information from specific compartments within the plant cell. The analysis of fatty acid groups, cell wall components, protein glycans, and starch, using both gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy are presented here. Fatty acid labeling determinations are sometimes highly convoluted. Derivatization to butyl amides reduces the errors in isotopomer resolution and quantification, resulting in better determination of fluxes into seed lipid reserves, including both plastidic and cytosolic reactions. While cell walls can account for a third or more of biomass in many seeds, no quantitative cell wall labeling measurements have been reported for plant flux analysis. Hydrolyzing cell wall and derivatizing sugars to the alditol acetates, provides novel labeling information and thereby can improve identification of flux through upper glycolytic intermediates of the cytosol. These strategies improve the quantification of key carbon fluxes in the compartmentalized flux network of plant cells.