Regional protein synthesis was investigated during severe, insulin-induced hypoglycemia in anesthetized, artificially ventilated rats. Three minutes after the cessation of spontaneous EEG activity, animals received a single injection of L-[3H]tyrosine and were killed 30 min later. Autoradiographs revealed an almost complete inhibition of amino acid incorporation in the cerebral cortex, hippocampus, and caudate-putamen of hypoglycemic rats. Thalamus and spinal cord were less affected, and in hypothalamus, cerebellum, and brainstem the pattern of [3H]tyrosine incorporation was identical in control and experimental animals. The areas showing severely reduced protein biosynthesis during hypoglycemia coincide with those exhibiting extensive breakdown of energy metabolism, reduced cerebral metabolic rate for glucose, and high susceptibility to persistent cell damage. High-resolution autoradiography employing tritiated amino acids permits the assessment of regionally impaired protein biosynthesis and may thus be useful for the identification of neuronal populations at risk during hypoglycemia of varying degrees and of those cells most likely to recover after glucose infusion.