Hlx is a homeobox transcription factor gene that is expressed in intestinal and hepatic mesenchyme of the developing mouse embryo and is essential for normal intestinal and hepatic development. Because of the morphological and molecular similarities in the development of the digestive system across species, we hypothesized that the Hlx gene and protein sequences and expression patterns would be conserved among vertebrates. Comparison of the Hlx gene orthologues of human, chimpanzee, mouse, rat, pufferfish (Fugu) and zebrafish demonstrates that these six genes share an identical organization with four exons and three introns. Comparison of the inferred Hlx protein sequences from these and three additional species (chick, Spanish ribbed newt and rainbow trout) reveals significant sequence identity, with identical homeodomains. The expression of Hlx in the mesenchyme of developing chick embryos is highly similar to that of mouse. Fugu Hlx is expressed in a tissue-specific manner that is similar though not identical to that of mouse, suggesting a conservation of Hlx function between mammals and birds. The mammalian and fish Hlx genes share a putative 5' upstream enhancer as well as an inverted repeat containing CCAAT boxes on opposite strands that we have previously shown to be important for mouse Hlx gene expression. These results suggest that the function of Hlx and the mechanisms regulating its expression are highly conserved in mammals, birds, amphibians and fish.