The establishment of retinal identity and the subsequent patterning of the optic vesicle are the key steps in early vertebrate eye development. To date little is known about the nature and interaction of the genes controlling these steps. So far few genes have been identified that, when over-expressed, can initiate ectopic eye formation. Of note is Six3, which is expressed exclusively in the anterior neural plate. However, 'loss of function' analysis has not been reported. Using medaka fish, we show that vertebrate Six3 is necessary for patterning of the anterior neuroectoderm including the retina anlage. Inactivation of Six3 function by morpholino knock-down results in the lack of forebrain and eyes. Corroborated by gain-of-function experiments, graded interference reveals an additional role of Six3 in the proximodistal patterning of the optic vesicle. During both processes of vertebrate eye formation, Six3 cooperates with Pax6.