In long-term culture of mouse bone marrow, the growth and differentiation of B-lineage lymphocytes depends on interaction with adherent cells or their products. The objectives of these studies were to characterize the types of cells present in the supporting adherent layer as well as the physical relationships of these cells with lymphocytes. With an extensive panel of antibodies against hemopoietic and lymphocyte antigens, two discrete nonlymphoid populations were identified: macrophages and undefined, large cells which we termed “stromal cells”. Lymphocyte clusters grew in actual contact with the latter cells only. Stromal cells lacked expression of most hemopoietic antigens, including the common leukocyte antigen, J11d, heat stable antigen (M1/69), Thy-1 and BP1. Antigens expressed by stromal cells were detected by AA4.1, our 94.2 antibody, and antibody to the Forsmann antigen, but the most distinguishing characteristics of the lymphocyte-binding stromal cells were production of basement membrane components, laminin and collagen IV, and the extremely low uptake of acetylated low density lipoprotein (LDL). Using acetylated LDL uptake as a sorting criterion, the lymphocyte-binding stromal cells were separated from the macrophages, recultured and shown to support lymphocyte proliferation. We found the binding between stromal cells and lymphocytes to be highly selective and dependent on divalent cations; hence, specialized adhesion mechanisms may have a role in B cell development. Moreover, our studies suggest that phosphatidylinositolanchored cell surface molecules may be involved in this adhesion. Our findings demonstrate the possibility that a single cell type provides physical support and proliferation stimuli for early B-lineage cells. This accessory cell is not a macrophage; rather, it has features of an endothelial or epithelial cell.