Neurolin-a and Neurolin-b (also called alcam and nlcam, respectively) are zebrafish orthologs of human ALCAM, an adhesion protein of the immunoglobulin superfamily with functions in axon growth and guidance. Within the developing zebrafish retina, onset and progression of Neurolin-a expression parallels the pattern of retinal ganglion cell (RGC) differentiation. By using a morpholino-based knockdown approach, we show that Neurolin-a (but not Neurolin-b) is necessary for a crucial step in RGC differentiation. Without Neurolin-a, a large proportion of RGCs fail to develop, and RGC axons are absent or reduced in number. Subsequently, Neurolin-a is required for RGC survival and for the differentiation of all other retinal neurons. Neurolin-b is expressed later in well-differentiated RGCs and is required for RGC axon pathfinding. Without Neurolin-b, RGC axons grow in highly aberrant routes along the optic tract and/or fail to reach the optic tectum. Thus, the zebrafish Neurolin paralogs are involved in distinct steps of retinotectal development. J. Comp. Neurol. 513:38–50, 2009. © 2008 Wiley-Liss, Inc.