Valid models are the foundation of systems biology. However, even well-established models may warrant reassessment. A testable feature of the currently accepted vacuolar model for fructan biosynthesis is its independence from metabolic energy at substrate level. The effects of limiting energy provision on fructan biosynthesis in grass leaves were determined. It was found that, in darkness in air, the rate of fructan accumulation was reduced to half relative to a light control. In darkness under anoxia the process was immediately abolished. In the light, the leaf sucrose concentration remained high, but in darkness +/- O(2), 40% of this sucrose was rapidly degraded. The constant rate of dark-aerobic fructan accumulation was independent of the decrease in sucrose concentration. Constant rates of aerobic fructan synthesis were independent of marked changes in extractable polymerase rates. In the dark under anoxia, fructan accumulation was abolished but leaves maintained > or = 80% of the extractable polymerase. Extractable polymerase rates cannot explain the rates of fructan accumulation observed in vivo, if the process is vacuolar. It was shown that the results were inconsistent with a vacuolar site for fructan synthesis. Six inhibitors of endomembrane function were shown to abolish fructan synthesis in vivo.