The tectobulbar tract is the first long-distance projecting fiber pathway to appear during the development of the avian optic tectum (dorsal half of the mesencephalon). Immunologically stained wholemounts of the E3 mesencephalon reveal that the early tectobulbar axons course in a dorsal-to-ventral direction and abruptly turn in a caudal direction shortly before reaching the ventral midline. During subsequent development, more tectobulbar axons are generated that form a parallel array of thick fascicles coursing ventrally within the mesencephalon. At this later stage of development, the tectobulbar tract bifurcates into an ipsilateral and contralateral pathway, both growing in a caudal direction near the mesencephalic ventral midline. Bifurcation and change in direction of growth is accompanied by a complete loss of the fasciculated growth pattern. Each tectobulbar axon is thus divided into a proximal fasciculated and a distal unfasciculated segment. Tectobulbar fascicles occupy the most superficial surface layer of the mesencephalon at early stages and are displaced deeper into the tissue beginning at embryonic day 5. In both of these locations, tectobulbar axons express molecules involved in axon-axon and axon-substrate interactions like the G4 antigen, neural cell adhesion molecule (N-CAM), neurofascin, and T61 antigen as revealed by immunohistochemistry and immunoblotting. Stripes of the mesencephalon explanted onto a basal lamina substratum show vigorous outgrowth of neurites. These processes grow in fascicles at a growth rate of 40 microns/h. Staining of the neurites with specific antibodies, as well as the position of the retrogradely labeled cell bodies, is in agreement with these processes being tectobulbar axons. This in vitro explant system was used to investigate the expression and possible functional involvement of N-CAM, neurofascin, G4 protein, and T61 antigen in the growth of these axons. The presence of antigen-binding fragments of polyclonal anti-G4 antibodies completely blocks fasciculation of the neurites but has no influence on their rate of elongation. Antibodies against N-CAM and neurofascin have no detectable effects. The number and length of the in vitro growing axons are reduced by the monoclonal T61 antibody. This effect is reversible. The elucidation of the exact course in vivo and the accessibility to the axons growing in vitro make the tectobulbar tract an excellent model system for the investigation of the role of these and other proteins in axonal growth and guidance during the development of the CNS.