Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and itsCaenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5 is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. We demonstrate that the Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, we show that corticospinal tract fibers respond differently to loss of UNC5H3. In addition, we observe corticospinal tract defects in mice homozygous for a spontaneous mutation that truncates the Dcctranscript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrateunc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates.