The mammalian circadian pacemaker of the suprachiasmatic nucleus (SCN) is entrained to the environmental light-dark cycle via a retinal projection, the retinohypothalamic tract (RHT). Several studies suggest that an excitatory amino acid, possibly glutamate, is involved in photic entrainment. However, it has not yet been established whether glutamate is a transmitter of the RHT itself. We have now identified terminals of the RHT in the SCN of brown Norwegian rats by intravitreous injections of horse radish peroxidase conjugated to cholera toxin. To detect glutamate immunoreactivity (IR), post-embedding immunocytochemistry was performed with polyclonal antibodies which were visualized for electron microscopy with colloidal gold particles. Retinal terminals had a significantly 82% higher glutamate-IR than their post-synaptic dendrites and a significantly 76% higher glutamate-IR than non-retinal terminals. These observations provide ultrastructural evidence that glutamate is a transmitter of the RHT.