Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo.