Extending axons are guided in part by diffusible chemoattractants that lure them to their targets and by diffusible chemorepellents that keep them away from nontarget regions. Floor plate cells at the ventral midline of the neural tube express a diffusible chemoattractant, netrin-1, that attracts a group of ventrally directed axons. Here we report that floor plate cells also have a long-range repulsive effect on a set of axons, trochlear motor axons, that grow dorsally away from the floor plate in vivo. COS cells secreting recombinant netrin-1 mimic this effect, suggesting that netrin-1 is a bifunctional guidance cue that simultaneously attracts some axons to the floor plate while steering others away. This bifunctionality of netrin-1 in vertebrates mirrors the dual actions of UNC-6, a C. elegans homolog of netrin-1, which is involved in guiding both dorsal and ventral migrations in the nematode.