Hyaluronic acid was localized in acetone-fixed cryostat sections of brain and spinal cord obtained from adult, newborn and embryonal rat. The sections were incubated with glial hyaluronate-binding protein (GHAP) of human origin and the protein was visualized by indirect immunofluorescence with monoclonal antibodies raised to human GHAP and not staining rat brain by immunofluorescence. GHAP is a brain extracellular matrix (ECM) glycoprotein, approximately 60,000 molecular weight, which is structurally related to the HA-binding region of cartilage ECM proteins. The distribution of hyaluronate in adult brain white matter and cerebellar cortex was similar to that previously reported for GHAP. In both cases, the reaction product formed a mesh surrounding myelinated axons and granule cells. Hyaluronate was also found in parts of the brain that did not contain GHAP. A finely reticulated mesh was observed in the neuropil between cell bodies in cerebral cortex and basal ganglia. Scattered cortical neurons were surrounded by a rim of reactive material. Perineural staining was the rule rather than the exception in spinal cord anterior horn motoneurons, inferior olivary nucleus, large bulbar reticular neurons and dentate nucleus of cerebellum. The only part of the brain which appeared relatively free of hyaluronate was the molecular layer of the cerebellum. In newborn and embryonal rat, the densely packed cell bodies in cerebral gray matter, periventricular germinal layer and external granular layer of cerebellum were surrounded by hyaluronate. Small droplets of hyaluronate were observed in between the cylindrical epithelial cells lining the neural tube in 11 day embryos. Non-myelinated fiber tracts and the molecular layer of the developing cerebellum were relatively unstained. No hyaluronate was detected in the ependyma lining the cerebral ventricles and the central canal of the spinal cord.