The distribution of glutamine synthetase was determined in rat brain by ultrastructural immunocytochemistry. Except for trace amounts in a rare indeterminate glial cell, all the reaction product was located in astrocytes. Specifically, none was observed in neurons, synaptic endings, oligodendrocytes, microglial cells, pericytes, endothelial cells and other mesenchymal vascular elements. The results of this study clearly indicate that the astrocyte forms the compartment in brain concerned with glutamine synthesis, thereby assigning a key role to the astrocyte in the metabolism of ammonia and the putative neurotransmitters, glutamic acid and gamma-aminobutyric acid. The localization of glutamine synthetase in astrocytes additionally provides a valuable marker for a number of neurobiological studies.