Following immunizations of BALB/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immunoprecipitates a Mr 105,000 glycoprotein on K562 cells. (b) Two MoAbs, 14B6 and 12B1, react with cells of the monocytic series. MoAb 14B6, which also faintly stains platelets, is reactive with immature myeloid cells and the majority of hematopoietic progenitors. The 14B6 antigen has been immunoprecipitated from 12-O-tetradecanoylphorbol-13-acetate treated K562 cells as a Mr 130,000-100,000 protein. Antigen 12B1 is expressed only on cultured monocyte/macrophages and is restricted to a subpopulation of monocytes and to follicular dendritic cells. It is not detected on hematopoietic progenitors. Immunoprecipitation experiments performed on 12-O-tetradecanoylphorbol-13-acetate treated K562 cells revealed a glycoprotein with a molecular weight of 93,000-86,000. (c) Two anti-K562 MoAbs, CF4 and HE10, recognize a myeloid differentiation antigen expressed from the granulomonocytic colony forming unit stage to polymorphonuclear neutrophils. These MoAbs detect an apparently original glycolipid moiety distinct from LeX. (d) Two MoAbs recognize antigens expressed on the granulomonocytic series. 2E1 recognizes the monocyte low affinity Fc receptor (Mr 40,000) and defines a new cluster of myeloid differentiation (CDw32). The antigen is expressed on a small portion of immature hematopoietic progenitors. 8F5 identifies a Mr 95,000 protein which is also present on plasma cells. In some experiments, it is detected on erythroid colony forming unit analysis. Immunizations with K562 cells thus resulted in the production of antibodies recognizing antigens of the monocytic, granulocytic, as well as erythroid series. However, three of them are also detected on hematopoietic progenitors.