To determine the role of the floor plate (FP) in CNS development, I have used labeling techniques, including immunolabeling, to analyze cyclops mutant embryos, which lack the FP. Except for the anterior brain, the mutant phenotype is almost exclusively confined to the vicinity of the ventral CNS midline. In the midbrain, the number of ventral neurons is reduced and cell patterning is disturbed. In contrast, the neuronal arrangement in the spinal cord is almost normal, including in particular both primary and secondary motoneurons. Longitudinal axonal bundles are disorganized in both the brain and spinal cord. Laser ablating the FP in wild-type embryos locally phenocopies cyclops axonal disturbances, and transplanting wild-type FP precursor cells into mutants locally rescues the disturbances. These results demonstrate a significant role for the FP in pathfinding and fasciculation by axons in situ, especially during their longitudinal courses.