Abstract An in vacuo micromethod is described, involving diffusion plating, for the determination of changes with time in the rates of formation and specific activities of the CO 2 produced by 50 mg. of tissue slices. Experiments were conducted in which the concentration and specific activity of the C 14-labeled glucose in the medium were kept rigidly constant throughout the entire course of the incubation period. The constancy in the tissue's environment was maintained by replacement of the incubation medium plus gas phase every 30–60 min. As little as 20–120 μl. CO 2, produced at each 20- to 60-min. interval, was measured with an accuracy of 5%. Measurements made after isotopic equilibrium of the respired CO 2 was attained showed that as much as 15% of this CO 2 produced by rat cerebral cortex slices was derived from a source or sources other than the labeled glucose, i.e., from a substrate or substrates within the slices.