The incorporation in vivo of radiocarbon from 14C-bicarbonate in blood into relevant metabolites in rat brain is described. The animals, partially hepatectomized and nephrectomized, received the tracer bicarbonate via the intravenous route. The time course of label was followed in CO2 of blood and brain, in the anionic and cationic fractions of brain extract, in aspartate, glutamate, glutamine and in free glucose and in glycogen. From the tracer kinetic data a flux of 0.08 microgram atom fixed carbon min-1.g-1 brain tissue was calculated. Substantial amounts of 14C were found in free glucose, only a few percent in glycogen. The flux of newly synthetized glucose was approximated to 0.5--1.0 percent of the steady state level of glucose in brain tissue. In special experiments the localization of 14C in the carbon chain of aspartate and glucose was examined. 5 min following the tracer injection a practically total randomization of 14C between C-1 and C-4 aspartate was seen. From the radioactivity in glucose 94 percent were found in C-3 and C-4, only 6 percent in residual carbon. This 14C-pattern is typical for the labelling of glucose by CO2 fixation and retrograde Embden-Meyerhof pathway.