Regeneration of optic axons in the continuously growing optic system of adult zebrafish was analyzed by anterograde tracing and correlated with the mRNA expression patterns of the recognition molecules ephrin-A2 and ephrin-A5b in retinal targets. The optic rectum and diencephalic targets are all reinnervated after a lesion. However, the rate of erroneous pathway choices was increased at the chiasm and the bifurcation between the ventral and dorsal brachium of the optic tract compared to unlesioned animals. Tracer application to different retinal positions revealed retinotopic reinnervation of the tectum within 4 weeks after the lesion. In situ hybridization analysis indicated the presence of rostral-low to caudal-high gradients of ephrin-A2 and ephrin-A5b mRNAs in unlesioned control tecta and after a unilateral optic nerve lesion. By contrast, the parvocellular superficial pretectal nucleus showed retinotopic organization of optic fibers but no detectable expression of ephrin-A2 and ephrin-A5b mRNAs. However, a row of cells delineating the terminal field of optic fibers in the dorsal part of the periventricular pretectal nucleus was intensely labeled for ephrin-A5b mRNA and may thus provide a stop signal for ingrowing axons. Ephrin-A2 and ephrin-A5b mRNAs were not detectable in the adult retina, despite their prominent expression during development. Thus, given a complementary receptor system in retinal ganglion cells, expression of ephrin-A2 and ephrin-A5b in primary targets of optic fibers in adult zebrafish may contribute to guidance of optic axons that are continuously added to the adult projection and of regenerating axons after optic nerve lesion. (C) 2000 Wiley-Liss, Inc.