The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein.