BALB/c mice were immunized with uninduced K562 erythroleukemia cells and hybridomas were isolated after fusion of immune spleen cells to P3/NS1 murine myeloma cells. One selected hybrid, designated 10L-30, secreted an antibody of subclass immunoglobulin G2a which was specific for hematopoietic cells. Analysis of 10L-30 binding by complement-mediated cytotoxicity, indirect immunofluorescence, solid-phase radioimmunoassay, and mixed hemadsorption assay indicated that the 10L-30 antigen was expressed on the myeloid cell lines K562, KG-1A, KG-1, some B- and T-lymphoid cell lines, and all normal human peripheral blood T-lymphocyte samples tested, but was absent on the more differentiated myeloid cell lines HL-60, ML-2, ML-3, and normal blood granulocytes. Induction of erythroid differentiation in hemin-treated K562 cells caused a 10-fold reduction in 10L-30 binding. Human erythroid and granulocytic progenitor cells, platelets, erythrocytes, and reticulocytes were nonreactive, as were a variety of nonhematopoietic human tumor cell lines. Freshly isolated leukemic bone marrow samples from patients with M5 (2 of 5), M6 (2 of 2), acute lymphoid leukemia (9 of 14), and chronic myeloid leukemia in lymphoid blast crisis (1 of 1) were 10L-30 positive. The combined evidence indicates that the 10L-30 antigen is a normal, hematopoietic-specific differentiation antigen which is strongly expressed on both immature cells of the myeloid lineage and more generally in lymphoid ontogeny. The 10L-30 antigen may be a useful marker of both normal and leukemic hematopoietic differentiation.