TheDrosophilagenesine oculis(so) is a nuclear homeoprotein that is required for eye development. Homologous genes toso,denotedSIXgenes, have been found in vertebrates. Among theSIXgenes,SIX3is considered to be the functional homologue ofso.To provide insight into the potential implications ofSIX3in human ocular malformations, we have cloned and characterized the humanSIX3gene. In human eye,SIX3produces a 3-kb transcript that codes for a 332-amino-acid polypeptide that is virtually identical to its mouse and chick homologues. Expression ofSIX3was detected in human embryos as early as 5–7 weeks of gestation and found to be maintained in the eye throughout the entire period of fetal development. At 20 weeks of gestation, expression ofSIX3in the human retina was detected in the ganglion cells and in cells of the inner nuclear layer. The humanSIX3gene spans 4.4 kb of genomic DNA and is split in two exons separated by a 1659-bp intron.SIX3was mapped to human chromosome 2p16–p21, between the genetic markers D2S119 and D2S288. Interestingly, the map position of humanSIX3overlaps the locations of two dominant disorders with ocular phenotypes that have been assigned to this chromosomal region, holoprosencephaly type 2 and Malattia Leventinese.