In the mammalian retina, multiple mechanisms are responsible for guiding retinal ganglion cell axons to the optic fissure. In the present study we have used time-lapse videomicroscopy to show that, within the center of the retinal neuroepithelium, growth cones use a scaffold of previously formed axons as a substrate for guidance. High magnification time-lapse videomicroscopy of normal growth cones in the midretina have shown that they have the ability to alter their shape from long, streamlined forms that hug other axons to more flattened forms that move between axons or neuroepithelial endfeet. In studies on the role of specific cell interactions in these events, Fab fragments against L1 and NCAM, administered either alone or in combination, were found to have dramatic and distinct effects on retinal ganglion cell growth cones. Anti-L1 Fab fragments severely disrupted radial growth cone orientation and rate of outgrowth. The anti-L1-treated growth cones initially stalled for 2 h, then changed direction and, thereafter, resumed an elongation rate twice as fast as in control preparations. By contrast, anti-NCAM Fab did not affect growth cone direction, but caused subsets of growth cones to speed up initially, then to dramatically increase in size, stall, and eventually halt. These results imply that L1 and NCAM play different roles in the promotion and direction of axon growth and, along with repulsive molecules and physical channels, provide essential information for the unidirectional growth of retinal axons into the optic fissure.