AbstractThe Pax-6 gene encodes a transcriptional master regulator involved in the development of the eye. The quail Pax-6 gene is expressed in the neuroretina from two promoters, P0 and P1, P0 being activated at the onset of neuronal differentiation. In this paper we have identified two regions in the quail Pax-6 gene 5 flanking sequences, located 6 and 2.5kbp upstream from the P0 promoter that, like the previously characterised intragenic enhancer (EP enhancer), function as neuroretina-specific enhancers whose activity is restricted to the P0 promoter. Moreover, the activity of these 5enhancers in embryonic neuroretina cells is weaker at day5 than at day7, like the EP enhancer, and parallels the level of expression of P0-initiated mRNAs. Footprinting experiments show that neuroretina-specific factors bind to these 5enhancer elements. In addition we show that these quail Pax-6 enhancer elements, as well as the P0 promoter, are structurally and functionally conserved in humans. These results strongly suggest that these enhancer elements may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo. Thus the complex regulation of the quail Pax-6 gene is also conserved in humans.