Six1, which belongs to the sine oculis homeobox (Six) protein family, is an evolutionarily conserved transcription factor found in diverse organisms ranging from flatworms to humans. Six1 is expressed in various tissues including the nervous system during ontogenesis and has been implicated in cell differentiation, morphogenesis, and organogenesis of the ganglia and sensory placodes. However, the molecular mechanisms by which Six1 influences these events at the transcriptional level remain largely unknown. In this study, we used ChIP-Display to discover genomic regions occupied in vivo by Six1 homeoprotein in the developing mouse embryo. To validate Six1 occupancy at each of Six1-bound regions, ChIP - Quantitative PCR was performed using locus-specific primers, and it showed robust enrichment of the Six1-bound sequences. To address their regulatory potential, each of the Six1-bound sequences was cloned into a reporter cassette containing beta-globin minimal promoter and lacZ gene and assayed for enhancer activity in transgenic mouse embryos. One of the novel sequences, which was designated Six1-bound Regulatory Element 1 (SRE1), was sufficient to activate lacZ reporter expression in the cranial and spinal ganglia. Comparative genomic analysis identified SRE1 sequences from a number of vertebrate phyla. Transgenic embryos carrying SRE1 sequences from human, chicken and frog showed reporter expression in a pattern similar to that of mouse SRE1, indicating their functional conservation. Through mutational analysis, we further showed that a conserved binding site matching the consensus for Six1/2/4/5 is required for the SRE1 regulatory activity. These data suggest that SRE1 is a functionally conserved transcriptional enhancer regulated by Six1.