Members of the Eph‐B family of receptors tyrosine kinase and their transmembrane ligands have been implicated in dorsoventral patterning of the vertebrate retinotectal projection. In the zebrafish retinotectal system, however, ephrinB2a is expressed strongly in the posterior tectum, in tectal neurons that form physical contacts with retinal ganglion cell (RGC) axons. In the gnarled mutant, where tectal neurons form ectopically in the pretectum, RGC axons stall before entering the tectum, or else are misrouted or branch aberrantly in the tectal neuropil. Ectopic expression of ephrinB2a in the anterior midbrain of wild‐type embryos, with the aid of baculovirus, also inhibits RGC axon entry into the tectum. In vitro, zebrafish RGC axons are repelled by stripes of purified ephrinB2a. It is proposed that ephrinB2a may signal a subpopulation of RGC axons that they have reached their target neurons in the tectum. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 57–65, 2004