We describe here a new murine homeo box gene, denoted Hlx, which is expressed within specific hematopoietic lineages. The cDNA sequence indicates that Hlx differs markedly from known vertebrate homeo box genes, and linkage analysis of an interspecific murine backcross showed that it resides at a novel homeo box locus on the distal portion of mouse chromosome 1. The Hlx homeo domain is most similar to that of the Drosophila H2.0 gene, but outside this region the two polypeptides are related only within a few short segments, the most notable being a motif (denoted Hep) also partially conserved in the engrailed and invected homeo proteins and possibly related to an octapeptide in certain paired box proteins. The presence of an intron within the Hlx homeo box at the same position as in several divergent Drosophila genes (H2.0, labial, Distal-less, proboscipedia, Abdominal-B, NK-1) suggests an ancient evolutionary relationship between these genes. RNA analysis of 67 murine hematopoietic cell lines and normal hematopoietic cells revealed Hlx expression throughout the myeloid/macrophage lineage and at early stages of B lymphocyte development but not in T lymphocytes, erythroid cells, or mast cells. Hence, Hlx is a candidate regulator of hematopoietic lineage commitment and maturation. It probably also functions outside the hematopoietic system, however, because Hlx mRNA could be detected in diverse adult tissues and in embryos from as early as day 8 of development.