EphA4 is a receptor tyrosine kinase of the Eph family previously designated Cek8 in chicken, Tyro1 in rat, and Sek1 in mouse, which is preferentially expressed in the embryonic and adult nervous system. We have mapped the distribution of EphA4 in the adult rat brain and spinal cord using a polyclonal antibody raised against a synthetic carboxy-terminal peptide. Immunoblotting experiments revealed that EphA4 is widely distributed in various regions of the adult rat brain. At the light microscopic level, intense immunoreactivity was apparent in the cerebral cortex, hippocampus, matrix compartment of the neostriatum, cholinergic neurons in the basal forebrain, cerebellar Purkinje cells, and substantia gelatinosa of the spinal cord. Among white matter tracts, EphA4 expression was detected in the corpus callosum, fornix, and posterior portion of the anterior commissure, but not in the lateral olfactory tract, mammillothalamic tract, or optic chiasm. Interestingly, expression in the optic chiasm is high at postnatal day 6, but decreases with the maturation of this structure. While in some regions of the neuropil neuronal cell bodies were prominently labeled, in others EphA4 immunoreactivity was detected in a punctate pattern. This punctuate staining did not coincide with synaptophysin localization. At the electron microscopic level, EphA4 immunoreactivity was observed in dendrites in the gray matter, particularly associated with dendritic spines, and in myelinated axons, but not their myelin sheaths in the white matter. The widespread distribution and diverse subcellular compartmentalization of EphA4 suggest that this receptor is important for the maintenance of multiple structures in the adult nervous system.