Free glycine is known to have vital functions in the mammalian brain, where it serves mainly as both neurotransmitter and neuromodulator. Despite its importance, little is known about the metabolic pathways of glycine synthesis and degradation in the central nervous system. In this study, the pathway of glycine metabolism in astroglia-rich primary cultures from rat brain was examined. The cells were allowed to degrade glycine in the presence of [U-14 C]glycine, [U-13 C]glycine or [ 15 N]glycine. The resulting intra-and extracellular metabolites were analyzed both by high-performance liquid chromatography and by 13 C/ 15 N nuclear magnetic resonance spectroscopy. Glycine was rapidly consumed in a process obeying first-order kinetics. The initial glycine consumption rate was 0.47 nmol per mg protein. The half-life of glycine radiolabel in the incubation medium was shorter than that of glycine mass. This suggests that glycine is produced from endogenous sources and released simultaneously with glycine uptake and metabolism. As the main metabolites of the glycine carbon skeleton in astroglia-rich primary cultures from rat brain, serine and lactate were released during glycine consumption. The main metabolite containing the glycine amino nitrogen was glutamine. To establish a metabolic pathway from glycine to serine in neural tissue, homogenates of rat brain and of neural primary cultures were assayed for their content of serine hydroxymethyltransferase (SHMT) and glycine cleavage system (GCS). SHMT activity was present in homogenates of rat brain as well as of astroglia-rich and neuron-rich primary cultures, whereas GCS activity was detectable only in homogenates of rat brain and astroglia-rich primary culture. Of the two known SHMT isoenzymes, only the mitochondrial form was found in rat brain homogenate. It is proposed that, in neural tissue, glycine is metabolized by the combined action of SHMT and the GCS. Owing to the absence of the GCS from neurons, astrocytes appear to be the only site of this part of glycine metabolism in brain. However, neurons are able to utilize as energy source the lactate formed by astroglial cells in this metabolic pathway.