The identities of neurotransmitters of the pretecto-olivary projection neurons and of the nerve terminals contacting them were investigated using a double-label method with retrograde labelling in combination with gamma-aminobutyric acid (GABA) and glutamate immunocytochemistry in the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic system in the rat both light and electron microscopically. At the light microscopic level, the somata of all projection neurons identified by a label of horseradish peroxidase reaction product were stained moderately but reliably for glutamate immunoreactivity. In no case, any retrogradely labelled neuron was found to be stained for GABA immunoreactivity. However, the somata and proximal dendrites of these cells were surrounded with many intensively stained puncta, indicating strong reactions with the anti-GABA antibodies. In contrast, no immunostaining with anti-glycine or anti-taurine antibodies was obtained. Electron microscopic investigations demonstrated that immunogold-positive axosomatic or axodendritic synapses on the retrogradely labelled neurons corresponded to some of the GABA-positive puncta in semithin sections. The results suggest that the projection neurons receive a strong inhibitory input mediated by GABA and send their directionally selective information to the inferior olive by glutamatergic projections.