There has been increasing interest in the involvement of mammalian homeobox (HOX) genes in hematopoietic regulation. The HOX genes are clustered in 4 chromosomes in mice and humans. In general, 5' end HOX gene expression is predominant in hematopoietic stem cell populations, whereas 3' end HOX gene expression are primarily found in committed progenitor cells. Furthermore, HOX genes of the A cluster are generally found in myelomonocytic cells, B cluster genes in erythropoietic cells, and C cluster genes in lymphoid cells. The results presented here concentrate on a single gene, namely HOX B6. Preliminary observations using whole mount in situ hybridization showed that both HOX B6 and erythropoietin (EPO) gene expression occurred in exactly the same areas of the 8.5-day mouse embryo. As a consequence, we studied the expression of HOX B6 and EPO gene expression from 6.5 to 19.5 days of gestation, in the neonate, and in the adult. It was found that the sequential transfer of erythropoiesis in different organs during development was followed by a similar transfer of HOX B6 and EPO gene expression. Between days 16.5 and 17.5, both HOX B6 and EPO gene expression decrease in the fetal liver, even though hepatic erythropoiesis continues to decline and is transferred to the fetal spleen. Precisely at this time point, HOX B6 and EPO gene expression are transferred to both the fetal spleen and fetal kidney. However, surprisingly, expression of both genes increases again in the fetal liver just before birth. HOX B6 is expressed in cells from in vitro erythropoietic colonies (colony-forming unit-erythroid and burst-forming unit-erythroid) and TER-119+ erythroid cells but not in hematopoietic or nonhematopoietic stem cell populations. When the latter two populations are allowed to differentiate into erythropoietic cells, HOX B6 and erythroid-relevant markers are expressed. The results indicate that HOX B6 is intimately involved in the regulation of the erythropoietic system and could be a marker for this lineage.