We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways.