The circadian pacemaker in the suprachiasmatic nuclei (SCN) is entrained to the environmental light-dark cycle via a direct retinal projection to the hypothalamus. This projection is thought to use glutamate or aspartate as neurotransmitter. 3H-D-Aspartate was microinjected in the SCN and adjacent hypothalamic nuclei of Syrian hamsters. This neuronal tracer is selectively taken up by terminals of neurons that use glutamate or aspartate as neurotransmitter and retrogradely transported to their perikarya. With autoradiography labeled cells were visualized in the retinal ganglion cell layer. Labeled cells were also found in a subset of brain nuclei known to project to the injection area. Labeled cells were detected in the bed nucleus of the stria terminalis, paraventricular nucleus of the thalamus, lateral septal nucleus, and medial amygdaloid nucleus. No labeled cells were observed in the medial septal nucleus, intergeniculate leaflet, and ventral lateral geniculate nucleus, which are also known to project to the SCN. Our results indicate that glutamatergic/aspartatergic retinal ganglion cells project to the SCN and adjacent medial hypothalamic nuclei. Moreover, the SCN may receive glutamatergic/aspartatergic input from the brain neuronal subsets that were retrogradely labeled with 3H-D-aspartate.