Pax6 is a critical transcription factor in the development of the eye, pancreas, and central nervous system. It is composed of two DNA-binding domains, the paired domain (PD), which has two helix-turn-helix (HTH) motifs, and the homeodomain (HD), made up from another HTH motif. Each HTH motif can bind to DNA separately or in combination with the others. We identified three novel binding sites that are specific for the PD and HD domains of human PAX6 from single-copy human genomic DNA libraries using cyclic amplification of protein binding sequences (CAPBS) and electrophoretic mobility shift assays (EMSAs). One of the binding sites was found within sequences of repetitive Alu elements. However, most of the Alu sequences were unable to bind to PAX6 because of a small number of mismatches (mostly in CpG dinucleotide hot spots) in the consensus Alu sequences. PAX6 binding Alu elements are found primarily in old and intermediate-aged Alu subfamilies. These data along with our previously identified B1-type Pax6 binding site showed that evolutionarily conserved Pax6 has target sites that are disparate in primates and rodents. This difference indicates that human and mouse Pax6-regulated gene networks may have evolved through these lineage-specific repeat elements.