L-Glutamic acid was investigated as the excitatory transmitter released from the optic nerve terminals of the pigeon [Columba livia] optic tectum. Superficial layers of the tectum contained high levels of endogenous glutamate and accumulated L-[3H]glutamate by a high affinity uptake process. Subcellular and autoradiographic studies indicated that 10-30% of the exogenously accumulated L-[3H]glutamate was localized within synaptosomes, and that 11-15% of the synaptosomes were labeled. The glutamate-accumulating synaptosomes sedimented to the same isopycnic density as pinched-off optic nerve terminals. GABA and noradrenaline-accumulating synaptosomes were also associated with this subcellular population. Retinal ablation did not change endogenous glutamate concentrations or the high affinity uptake of glutamate. L-Glutamate was discussed as the optic nerve transmitter and in the context of previous evidence implicating glutamate as an excitatory transmitter.