Homeodomain containing transcription factors serve important functions in patterning the embryo during vertebrate development. We have isolated cDNA clones encoding a novel protein, named Alx-4, that contains a paired-type homeodomain. Analysis of the homeodomain sequence shows that Alx-4 belongs to a family of genes that are related to the Drosophila gene aristaless, and includes the mammalian genes Alx3, Cart-1, MHox, and S8. We have analyzed the expression of Alx-4 during development by Northern blot and whole mount in situ hybridization. In addition, we have generated antibodies to recombinant Alx-4 protein and identified Alx-4 protein in nuclear extracts prepared from mouse embryos. The expression pattern of Alx-4 suggests that it may play a role in the patterning of structures derived from craniofacial mesenchyme, the first branchial arch, and the limb bud. Our results provide a starting point for the analysis of a new member of the family of paired type homeodomain proteins.