Netrin-1 has profound in vitro effects on the growth properties of vertebrate embryonic axons. In addition, netrin-1 mRNA is found in the floor plate of the embryonic nervous system, an intermediate target of many axons, including commissural axons that are affected by netrin-1 in vitro. Moreover, genetic studies of netrin-1 homologs in Caenorhabditis elegans and Drosophila implicate these proteins in commissure formation. We raised polyclonal antisera that recognize chick netrin-1 in fixed tissue sections. The antisera were used to immunohistochemically map netrin-1 in the embryonic spinal cord, brain, and retina. The relationship between netrin-1 localization and the growth of pioneering axons suggests roles for netrin-1 in the regulation of circumferential, commissural, and longitudinal axon growth in the spinal cord and brain. The data also suggest that the primary or sole effect of netrin-1 on pioneering spinal cord commissural axons is haptotactic. Furthermore, the pattern of netrin-1 localization raises the possibility that this protein helps mediate neuronal migration in the spinal cord, brain, and retina.