Vsx-1 and Vsx-2 are two homeobox genes that were cloned originally from an adult goldfish retinal library. They are members of the paired-like:CVC gene family, which is characterized by the presence of a paired homeodomain and an additional conserved region, termed the CVC domain. To analyze the possible roles for Vsx-1 and Vsx-2 in eye development, we used in situ hybridization to examine their expression patterns in zebrafish and goldfish embryos. Vsx-2 is initially expressed by proliferating neuroepithelial cells of the presumptive neural retina, then it is down-regulated as differentiation begins, and it is finally reexpressed at later stages of differentiation in a subset of cells, presumed to be bipolar cells, in the inner nuclear layer. In contrast, Vsx-1 is expressed only weakly in undifferentiated, presumptive neural retina and is then up-regulated selectively in presumptive bipolar cells at early stages of differentiation (when Vsx-2 is turned off), before decreasing to an intermediate level, which is maintained in the differentiated (adult) retina. The restricted expression patterns of Vsx-2 correspond to the observed phenotypes in mice with the ocular retardation mutation (orJ), further supporting the notion that Vsx-2 and Chx10 are homologues. The sequential complimentary and then corresponding expression patterns of Vsx-1 and Vsx-2 suggest that these similar transcription factors may be recruited for partially overlapping, but distinct, functions during the development of the retina.