Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.Brassica napus (rapeseed, canola) is one of the world's major oilseed crops and is also a well studied model for oilseed metabolism (1-21). The main storage compounds in seeds of B. napus are oil (triacylglycerols) and storage proteins, which are derived from sugars and amino acids taken up from the surrounding endosperm liquid (11,12,22,23). Because of its high oil content and ease of genetic transformation, B. napus has also been a target for metabolic engineering of oil metabolism. However, some attempts to engineer plant oils have had limited success (for a review, see Ref. 24). In order to make advances in improving oil yield and quality, a more detailed understanding of metabolism during seed development is needed. In particular, a number of fundamental metabolic issues remain unresolved. These include the source(s) of reductant and ATP for fatty acid synthesis; the degree to which cytosolic, plastidial, and mitochondrial metabolic fluxes are integrated; and the chief metabolic and transport route(s) by which carbon flows from maternal sources to seed storage products.