In the developing nervous system, motor axons grow away from the ventral midline floor plate, suggesting that the latter might be a source of repulsive axonal guidance cues. In donor to host transplantation experiments, ectopic pieces of floor plate were positioned between chick hindbrain motor neurons and their exit points. Immunohistochemistry and retrograde axonal labeling techniques demonstrated that motor axons diverted from their normal pathways to avoid grafted floor plate, often traversing abnormally long circuitous trajectories to reach exit points. When ventral explants of rat hindbrain and spinal cord were cocultured at a distance from floor plate explants within collagen gel matrices, the outgrowth of motor axons was dramatically reduced from explant borders that faced the floor plate. Thus, the floor plate secretes diffusible repulsive cues in vitro that may exclude motor axons from the midline during development.