Evidence has been presented in recent years that support the hypothesis thatN-acetylaspartylglutamate (NAAG) may be involved in synaptic transmission in the optic tract of mammals. Using a modified fixation protocol, we have determined the detailed distribution of NAAG immunoreactivity (NAAG-IR) in retinal ganglion cells and optic projections of the rat. Following optic nerve transection, dramatic losses of NAAG-IR were observed in the neuropil of all retinal target zones including the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, the dorsal and medial terminal nuclei and suprachiasmatic nucleus. Brain regions were microdissected and NAAG levels measured by a radioimmunoassay (RIA) (IC₅₀:NAAG= 2.5nM,NAA= 100 μM;smallest detectable amount= 1–2pg/assay). decreases (50–60%) in NAAG levels were detected in the lateral geniculate, superior colliculus and suprachiasmatic nucleus. Moderate losses (25–45%) were noted in the pretectal nucleus and the nucleus of the optic tract. Smaller changes (15–20%) were detected in the paraventricular nucleus and the pretectal area. These results are consistent with a synaptic communication role for NAAG in the visual system.