Bilaterally symmetric animals must be capable of transmitting information between the left and right sides of their body to integrate sensory input and to coordinate motor control. Thus, many neurons in the central nervous system (CNS) of a wide variety of higher organisms project so-called commissural axons across the midline. Interestingly, these axons are never observed to re-cross the midline. On the other hand, some neurons project axons that remain on their own (ipsilateral) side of the CNS, without ever crossing the midline. Recent studies demonstrate that specialized cells which reside at the ventral midline of the developing vertebrate spinal cord and Drosophila ventral nerve cord play critical roles in regulating the guidance of both crossing and non-crossing axons. For example, these cells secrete positively-acting guidance cues that attract commissural axons over long distances to the midline of the CNS. Furthermore, short-range interactions between guidance cues present on the surfaces of midline cells, and their receptors expressed on the surfaces of pathfinding axons, allow commissural axons to cross the midline and prevent ipsilaterally projecting axons from entering the midline. Remarkably, as commissural axons cross over to the opposite side of the CNS, the molecular composition of their surfaces is dynamically altered so that they become responsive to repulsive midline guidance cues that they had previously ignored. Thus, this exquisitely controlled guidance system prevents commissural axons from crossing the midline more than once. Strikingly, many of the molecular mechanisms that control midline guidance appear to be evolutionarily conserved.