A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.