The Roundabout (Robo) family of receptors and their Slit ligands play well-established roles in axonal guidance, including in humans where horizontal gaze palsy with progressive scoliosis (HGPPS) is caused by mutations in the robo3 gene. Although significant progress has been made toward understanding the mechanism by which Robo receptors establish commissural projections in the central nervous system, less is known about how these projections contribute to neural circuits mediating behavior. In this study, we report cloning of the zebrafish behavioral mutant twitch twice and show that twitch twice encodes robo3. We show that in mutant hindbrains the axons of an identified pair of neurons, the Mauthner cells, fail to cross the midline. The Mauthner neurons are essential for the startle response, and in twitch twice/robo3 mutants misguidance of the Mauthner axons results in a unidirectional startle response. Moreover, we show that twitch twice mutants exhibit normal visual acuity but display defects in horizontal eye movements, suggesting a specific and critical role for twitch twice/robo3 in sensory-guided behavior.