We have identified and mapped three members of a new family of vertebrate genes, designated Eya1, Eya2 and Eya3, which share high sequence similarity with the Drosophila eyes absent (eya) gene. Comparison of all three murine Eya gene products and that encoded by the Drosophila eya gene defines a 271 amino acid carboxyl terminal Eya domain, which has been highly conserved during evolution. Eya1 and Eya2, which are closely related, are extensively expressed in cranial placodes, in the branchial arches and CNS and in complementary or over-lapping patterns during organogenesis. Eya3 is also expressed in the branchial arches and CNS, but lacks cranial placode expression. All three Eya genes are expressed in the developing eye. Eya1 is expressed in developing anterior chamber structures, including the lens placode, the iris and ciliary region and the prospective corneal ectoderm. Eya1 is also expressed in retinal pigment epithelium and optic nerve. Eya2 is expressed in neural retina, sclera and optic nerve sheath. Moreover, Eya1 and Eya2 expressions in the lens and nasal placode overlap with and depend upon expression of Pax6. The high sequence similarity with Drosophila eya, the conserved developmental expression of Eya genes in the eye and the Pax6 dependence of Eya expression in the lens and nasal placode indicates that these genes likely represent functional homologues of the Drosophila eya gene. These results suggest that members of the Eya gene family play critical roles down-stream of Pax genes in specifying placodal identity and support the idea that despite enormous morphological differences, the early development of insect and mammalian eyes is controlled by a conserved regulatory hierarchy.