In order to determine the CNS distribution of the 57 kDa neural intermediate filament protein (NIFP), a specific antiserum was used in immunofluorescence studies on serial sections taken from each spinal cord level and at 300-microns intervals through the rat brain. The labeling pattern was recorded onto camera lucida tracings of adjacent sections stained with hematoxylin and eosin/luxol fast blue. Three major immunolocalization patterns were revealed. (1) Both large-caliber and fine-caliber axons of optic and all brain stem cranial nerves and their tracts except for the auditory portion of the VIIIth nerve. (2) An extensive array of fine-caliber fibers in the cerebellar white matter and brain stem with region-specific variation in pattern and density. Prominent among the regions with dense arrays of labeled processes were selective cerebellar afferent systems, particularly olivocerebellar fibers, visual afferents arising in the retina, and selective regions of reticular formation. In contrast to the brain stem, the telencephalon contained rare label. (3) Filamentous labeling of neuronal cytokarya in sensory ganglia and a small number of CNS nuclear groups, including all autonomic nuclei and the cholinergic pontine nuclei. On sections of selected CNS regions, distribution patterns of the 57 kDa NIFP were correlated to results obtained by in situ hybridization of a cDNA probe for the 57 kDa NIFP. The data suggest the possible existence of neuronal IF proteins specifically related to selective anatomic and/or neurotransmitter systems.