Selective uptake and bidirectional axonal migration of tritiated D-aspartate ([3H]d-asp) within a sub-population of retino-tectal neurons was shown by radioautography in the pigeon visual system. Six hours after either pulse injection or prolonged topical application of [3H]d-asp to the optic tectum, light microscope radioautographs exhibited an intense and preferential labeling of incoming optic fibers, within tectal layer 1. These axons could be traced back to their perikarya in the ganglion cell layer of the contralateral retina. Conversely, 6-48 h after intra-vitreous injection of [3H]d-asp, anterogradely-labeled axons could be followed from the fiber layer of the retina to layer 1-7 of the contralateral optic tectum. In addition, several mesodiencephalic primary visual relay nuclei showed some degree of radioautographic labeling. Labeled retinofugal axons originated from a restricted contingent of small intensely reactive ganglion cells which were heterogeneously distributed throughout the retina. These cells were particularly numerous in the postero-superior quadrant which is known to project to the ventro-caudal aspect of the tectum. Indeed, both radioautography and liquid scintillation measurements showed that the bulk of anterogradely transported radioactivity (more than 80% as free asp) was accumulated in this tectal region. In contrast, when [14C]l-leucine was injected together with [3H]d-asp, anterogradely transported 14c-labeled proteins were evenly distributed throughout the tectum. Finally, part of the radioactivity present in tectum 48 h after intraocular [3H]d-asp could be released 'in vitro' by 47 mm K + stimulation, in a Ca2+ dependent manner. These results strongly suggest that the pigeon retinal ganglion cells which are here reported selectively to take up, transport and release [3H]d-asp may well utilize l-aspartate, l-glutamate or a closely related substance as a neurotransmitter.