We identified five cDNA clones of the Six gene family which are expressed in retina. They are Six2, Six3 alpha and Six3 beta (which are derived from alternative splicing forms), Six5, and AREC3/Six4. All of these Six family genes possess extensive sequence similarity among each other in the so-homologous region (Six domain and homeodomain) but differ greatly in structure in some other regions. The amino acid sequence similarity of the so-homologous region to the previously identified AREC3/Six4 is 70.1% for Six2, 57.3% for Six3 alpha and Six3 beta, and 70.3% for Six5. The expression of these genes was observed in inner and outer nuclear layer, ganglion cell layer, and pigment epithelium of mouse retina by in situ hybridization. The so-homologous region of each Six family protein has specific DNA binding activity. Six5 and Six2 bind to the same sequence as does AREC3/Six4, while Six3 does not. These observations suggest that some of the Six family genes can regulate the same target genes.