Although it is well established that homeobox (HOX) genes play a key role in normal human embryogenesis, the expression and function of HOX genes in normal hematopoiesis is largely unknown. We have investigated by reverse transcriptase-polymerase chain reaction the mRNA expression of HOXB cluster genes (32 to 52 position in the cluster: from HOXB2 through B9) in 72% to 88% purified hematopoietic progenitor cells (HPCs) from adult peripheral blood induced in liquid suspension culture to gradual erythroid or granulopoietic (largely eosinophilic) differentiation and maturation by differential growth factor (GF) stimulus (ie, low-dose interleukin-3 [IL-3] and granulocyte-macrophage colony-stimulating factor [GM-CSF] and high-dose erythropoietin, or saturating amounts of IL-3/GM-CSF, respectively). Only B3 is expressed in quiescent HPCs. After GF treatment B3 expression is enhanced in the initial 24 hours and then through differentiation and maturation in erythroid and granulopoietic cultures. HOXB4 and B5 are induced at slightly later times and expressed through maturation in both lineages, whereas B6 is selectively induced in granulocytic differentiation. B2 is transiently expressed at low level in the granulopoietic pathway, whereas it is detected only in advanced stages of erythropoiesis: B7, B8, and B9 are essentially not detected. Functional studies were performed with antisense phosphorothioate oligomers to HOX mRNAs and included control analysis of the targeted mRNA. The results are strictly coherent with the HOX mRNA expression pattern: (1) anti-B3 oligomer (alpha-B3) treatment of purified HPCs induces a striking blockade of both erythroid and granulomonocytic colony formation (similarly, alpha-B3 treatment of K562 cell line causes a significant dose-related inhibition of cell proliferation); (2) alpha-B6 selectively and markedly inhibits granulomonocytic colony formation; (3) alpha-B4 and alpha-B5 cause a significant, less pronounced decrease of both colony types; (4) finally, alpha-B2 and alpha-B7, -B9 exert little and no effect, respectively. These studies provide novel evidence on the coordinate expression of selected HOXB cluster genes in erythropoiesis and granulopoiesis, particularly in the early stages of differentiation: B3 apparently functions as a master gene in early hematopoiesis, whereas B6 exerts a key selective function in the granulopoietic pathway.