Homologues of the homeobox genes sine oculis (so) and eyeless (ey) are important regulators of eye development in both vertebrates and invertebrates. A Drosophila paralogue of so, optix, is an orthologue of the vertebrate Six3 gene family. Our analysis of zebrafish six3.1 demonstrated retinal expression in two separate cell layers and the ciliary marginal zone. This pattern is consistent with the observations of Six3 in other vertebrates and indicates functional conservation. We studied the 5(') flanking region of six3.1 and showed that separate enhancing elements are required for expression at different stages of eye development. This analysis also revealed specific binding of zebrafish Pax6.1 protein to an element required for six3.1 expression in ganglion cells. Furthermore, an enhancement of six3.1 transcription by Pax6.1 was observed by co-injection experiments. These results provide evidence for a direct regulatory interaction between vertebrate Pax6 and Six3 genes in eye development.