Commissureless (Comm) is a key regulator of axon guidance at the midline of the Drosophila CNS. Here we report results from mosaic analysis experiments that demonstrate a cell-autonomous, neuronal requirement for Comm. Using the MARCM system to positively label clonal patches, we find that neurons which are not deficient for Comm function make commissural projections 85% of the time. Clones of neurons that are homozygous mutant for comm make commissural projections at a statistically significant reduced frequency: 70% for comm(1), a hypomorphic mutation, and 58% for comm(5), a near null mutation. These data suggest that commissural axon guidance is dependent upon regulated expression of Comm in neurons.