The establishment of polarity is an important step during organ development. We assign a function for the paired and homeodomain transcription factor Pax6 in axis formation in the retina. Pax6 is a key factor of the highly conserved genetic network implicated in directing the initial phases of eye development. We recently demonstrated that Pax6 is also essential for later aspects of eye development, such as lens formation and retinogenesis. In this study, we present evidence that a highly conserved intronic enhancer, alpha, in the Pax6 gene is essential for the establishment of a distal(high)-proximal(low) gradient of Pax6 activity in the retina. In the mature retina, the activity mediated by the alpha-enhancer defines a population of retinal ganglion cells that project to two sickle-shaped domains in the superior colliculus and lateral geniculate nucleus. Deletion of the alpha-enhancer in vivo revealed that retinal Pax6 expression is regulated in two complementary topographic domains. We found that Pax6 activity is required for the establishment, as well as the maintenance of dorsal and nasotemporal characteristics in the optic vesicle and, later, the optic cup.