The isthmo-optic nucleus of the centrifugal system in birds receives primarily input from the ipsilateral tectum and projects to the contralateral retina. The present study using brain slices and microiontophoresis shows that synaptic transmission from the tectum to the centrifugal nucleus in pigeons is excitatory. About 75% of tecto-isthmo-optic fibers are glutamatergic, mediated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid but not N-methyl-D-aspartate-receptors, and 25% of others may use nitric oxide as a transmitter or modulator. On the other hand, about 60% of isthmo-optic cells receive glutamatergic afferents, 20% receive nitric oxidergic afferents, and 20% of others receive both glutamatergic and nitric oxidergic afferents from the tectum. In the last group, it is more likely that both glutamate and nitric oxide may co-release from the same tecto-isthmo-optic terminals. All the isthmo-optic cells examined in the present study also receive γ-aminobutyric acid (GABA)ergic afferents via GABA A and GABA B receptors probably from some extratectal structures.