We have used retrograde transport and immunohistochemistry to study glutamate, aspartate, and enkephalin-like immunoreactive pathways from the mammillary nuclei to the anterior nuclei of the thalamus. Injections of wheat germ agglutinin conjugated to horseradish peroxidase into the anterodorsal thalamic nucleus resulted in retrogradely labelled cell bodies in the lateral mammillary nucleus, bilaterally, whereas injections into the anteroventral thalamic nucleus resulted in retrogradely labelled neurons in the ipsilateral medial mammillary nucleus. In three parallel series of sections immunoreacted for glutamate, aspartate, and enkephalin, respectively, 50-60% of the retrogradely labelled cell bodies were also immunolabelled for glutamate, 50-60% for aspartate, and 40-50% for enkephalin. The enkephalin-immunoreactive neurons may coincide with or constitute a separate population from the glutamate/aspartate-containing neurons. These results are compatible with the possibility that mammillothalamic projection neurons may use glutamate and/or aspartate and enkephalin as neurotransmitters.