This report describes the characterization and expression of a human myeloid differentiation antigen defined by use of an IgG1 monoclonal antibody (AHN-7). This antibody binds to many granulocytic precursors in normal marrow, to most but not all granulocyte-macrophage progenitors (CFU-GM), and to approximately half of nonlymphoid leukemia specimens. The protein antigens recognized by AHN-7 were purified from 35S-labelled HL-60 cells by antibody affinity column chromatography. The molecule reacting with AHN-7 was markedly heterogeneous, appearing as several forms ranging in pI from 4.5 to 6.4 with apparent molecular weights from 43,000 to 68,000. The molecules were not disulfide-linked. Proteins bearing the antigen were minor components of the plasma membrane. The antigen was expressed by normal human peripheral blood neutrophils, monocytes, eosinophils, and basophils, and weakly on a small percentage of lymphocytes; it was not detected in red blood cells, platelets, or the majority of lymphocytes. The antibody also bound to a variety of human myeloid leukemia cell lines but not to any lymphoid leukemia cell line tested. AHN-7 had no effect on several in vitro neutrophil functions tested.