Direct and indirect evidence strongly indicates that the proto-oncogene c-myb plays an important role in the regulation of both the proliferation and differentiation of hematopoietic cells. In addition, recent data suggest that the structurally related B-myb gene is also necessary for the proliferation of these cells. To help understand the relationship between these two related gene products during proliferation and differentiation of myeloid cells, we have studied in parallel the regulated expression of c-myb and B-myb RNAs and proteins in human myeloid cells that were either growth-arrested or induced to differentiate along different pathways. For this purpose, we have produced a polyclonal antibody directed against a fragment of the recombinant B-myb protein. We have thus been able to detect the B-myb protein in human cell lines and have found it to be a 93-kD protein localized in the nucleus. We have chosen two models to study the expression of both c-myb and B-myb mRNAs and proteins during myeloid proliferation and differentiation. One of the models was the HL-60 cell line, which can be induced to differentiate towards the monocytic pathway with either phorbol ester (phorbol myristate acetate) or vitamin D3 and towards the granulocytic pathway with either dimethyl sulfoxide or retinoic acid. In addition, we have studied another recently established human leukemic cell line, called GF-D8, which is strictly dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation. The results show that the expression of B- myb RNA and protein closely correlates with proliferation in all experimental setups studied, whereas the c-myb protein levels do not always do so. We observed that the c-myb protein levels decreased well before the decrease of B-myb protein and of proliferation itself during differentiation toward monocytes. Such a difference was not present during granulocytic differentiation, in which c-myb levels decreased, if anything, later than those of B-myb and proliferation. Most striking was the finding that high levels of c-myb RNA and protein, but not of B- myb, were present in the GF-D8 cell line, even after growth arrest by GM-CSF deprivation. These data suggest that B-myb may function solely in the regulation of cellular proliferation, whereas c-myb has additional functions, for example, in the maintenance of an undifferentiated state.