A simple and cheap one-step enzymatic method has been developed for the determination of 1-14C-glucose in plasma. C-1 of glucose is cleaved off as CO2 by treatment with hexokinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconic dehydrogenase. True 1-14C-glucose activity is then calculated as the difference between total radioactivity and radioactivity remaining after enzyme treatment and evaporation. The reaction is shown to be quantitative and specific, thus eliminating both labelled metabolites and label recycled to other positions in glucose. Two different types of pig experiments show that 1-14C-glucose, when determined by this method, is as irreversible a tracer as the commonly used 3-3H-glucose.