Neuronal growth cones establish appropriate connections with their targets during development by responding to both positive and negative guidance cues. The importance of repulsive and inhibitory cues in pathfinding and target selection has now been firmly established at the cellular and molecular levels. Observations in vitro have demonstrated developmentally significant repulsive interactions among various neuronal populations, providing the basis for molecular and functional characterization of several families of molecules that can mediate these guidance events. Analysis of both the expression and function of these molecules in vivo suggests how they, together with positive guidance cues, participate in the dynamic process of growth-cone guidance during both development and axonal regeneration.