Cell surface glycoproteins expressed on growth cones and axons during brain development have been postulated to be involved in the cell-cell interactions that guide axons into their target area. Nevertheless, an unequivocal description of the mechanism by which such molecules exert control over the pathway of a growing axon has not been done. As a crucial requirement in support of a relevant involvement of an axonal surface molecule in growth cone guidance, this molecule should be expressed in the growth cone. The developing retinotectal system provides an excellent opportunity to test whether a particular neuronal surface molecule fulfills the requirement of the spatiotemporal coincidence between its appearance and the emergence of growth cones because its setup follows the rule of chronotopy, i.e., the position of axons in a certain site is determined by the time of their arrival. We have analyzed axonin-1 and the neuron-glia cell adhesion molecule (Ng-CAM), two axonal surface molecules that promote neurite growth in vitro, for their expression in the retina and in the retinotectal system of the chick throughout its development. At stage 18, both axonin-like (A-LI) and Ng-CAM-like immunoreactivity (Ng-CAM-LI) are clearly present in the area where first retinal ganglion cells (RGCs) are generated. The immunoreactivity spreads synchronously with the formation of RGCs over the developing retina. From stage 32 on, the inner plexiform layer is also stained according to its temporospatial gradient of maturation. In later stages, the outer plexiform layer and the inner segments of photoreceptors also show immunoreactivity. The development of A-LI and Ng-CAM-LI along the optic nerve, chiasm, optic tract, and in the superficial layers of the optic tectum follows the chronotopic pattern of axons, as was found by earlier morphological investigations. Older axons loose their A-LI. This allows to localize the position of newly formed axons. The fact that A-LI and Ng-CAM-LI parallel the formation and maturation of axons suggests that axonin-1 and Ng-CAM may play an important role in the organization of the retinotectal system.