Biochemical and electrophysiological studies of mammalian CNS indicate that the amino-acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [3H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors.