N-Acetyl-aspartate is found in high concentrations in all areas of the brain, but is undetectable in non-neuronal tissue. In order to characterize the cellular localization of N-acetyl-aspartate in brain, highly specific monoclonal antibodies against N-acetyl-aspartate were produced by fusing spleen lymphocytes obtained from mice immunized with N-acetyl-aspartate conjugated to thyroglobulin by carbodiimide with P3/x63-Ag8.653 mouse myeloma cells. Clones were selected which secrete IgG2a(k) antibodies highly specific for conjugated N-acetyl-aspartate. Only 3-6% cross-reactivity with conjugated N-acetyl-aspartate-glutamate was observed at high antibody concentrations, whereas no cross-reactivity (less than 1%) was observed with conjugated N-acetyl-glutamate or aspartate. Preincubation of the antibodies with 0.5 mg/ml conjugated N-acetyl-aspartate blocked immunoreactivity more than 90%, while preincubation with conjugated N-acetyl-aspartate-glutamate and free N-acetyl-aspartate had no effect. Immunocytochemical staining has shown that N-acetyl-aspartate-like immunoreactivity is localized in neurons, which are widely distributed throughout the brain. The immunoreactive neurons exhibited intense staining of the perikarya, proximal dendrites and axons. No consistent pattern of distribution of immunoreactivity was observed with regard to primary neurotransmitter characteristics of stained neurons although neurons with long projections or extensive arbors, such as pyramidal cells in cortex, locus coeruleus, motor neurons and Purkinje cells, stained much more intensively than local circuit neurons.