A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and d-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.