The antigen expression of immature erythroid bone marrow cells was studied using two recently generated monoclonal antibodies (mAb), mAb 67A4 and 9C4, with specificities for the epithelial cell adhesion molecule E-cadherin (E-cad; mAb 67A4), and a novel 110 kDa differentiation antigen (mAb 9C4) with unknown molecular structure. Pappenheim staining of FACS-purified cells labeled with mAb 9C4 and anti-glycophorin A (GA) revealed that the majority of the 9C4+GA- and 9C4+GA+ cells consisted of erythroblasts. In contrast, the E-cad-positive population comprised normoblasts and erythroblasts. While the E-cad+GA- fraction contained mainly erythroblasts and basophilic normoblasts, the E-Cad+GA+ population was enriched in orthochromatic and polychromatophilic normoblasts. By colony assays of affinity column-purified cells it could be shown that erythroid colony forming units (CFU-E) were enriched and erythroid burst forming units (BFU-E) were depleted in the 9C4- and E-cad-positive fractions. Flow cytometric analysis of bone marrow cells double-labeled with mAb 67A4 and anti-CD71, anti-CD117, anti-CD34, or anti-GA revealed that about 90% of the E-cad-positive cells coexpressed CD71, about 70% were positive for CD117, about 50% for GA, and only about 5% coexpressed CD34. The expression pattern of 9C4 antigen was similar to that of E-Cad with the exception that only a minority of the 9C4-positive cells coexpressed GA. Lymphoid and myeloid markers were negative on both the E-Cad- and 9C4-positive populations. In these studies we describe the identification of a new mAb-defined antigen which is specifically expressed on erythroblasts and CFU-E(9C4) and demonstrate that E-Cad is not only expressed on epithelial cells but also on erythropoietic cells of defined maturational stages.