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This report describes studies carried out in an attempt t o define the antibody specificity of a rabbit antiserum generated t o the RPMl8226 line of human myeloma cells and then isolate and characterize the antigen. The antiserum reacted t o a significantly higher t i t e r in a quantitative semi-micro comp- The generation of a rabbit antiserum to a tissue culture line of human myeloma cells (RPMI 8226) and its reactivity with a number of different types of cells, including bone-marrow cells from normal individuals and patients with multiple myeloma, were described in previous reports (Staneck et al., 1974; Krueger et a/., 1976). The data suggested that the antibodies in the serum reacted with an antigen(s) only present o n human myeloma cells (i.e., malignant human plasma cells). O n the basis of this reactivity it was suggested that these cells possess a tumorspecific antigen. Recently, MacKenzie and Paglierone (1977) published data that also suggested that human myeloma plasma cells possessed a tumorspecific antigen(s). These authors showed that bonemarrow plasma cells from myeloma patients stimulated autologous and allogeneic peripheral blood lymphocytes (PBL) from myeloma patients in mixed leukocyte culture. Further, an isolated antigen preparation from myeloma plasma cells elicited proliferative and cytotoxic responses with lymphocytes from myeloma patients but not from nonmyeloma patients.The report describes studies carried out to define the antibody specificity of a rabbit antiserum generated to human myeloma cells as well as the results of our initial studies on the solubilization and isolation of the antigens of a line of human myeloma cells. MATERIAL A N D MtTHODS C d l cultureThe RPMI 8226 cell line was obtained from Dr. George Moore, Denver General Hospital, Denver, Colorado. Cells wcre cultured in RPMI 1640 medium supplemented with 20 heat-inactivated fetal calf serum and 0.4 % glutamine at 37" C in a 5 % C0,-95% air environment. The RPMl 4098 cell line (normal human lymphocytes) was obtained from Associated Biomedic Systems, Inc., Buffalo, New York and grown in Dulbecco's modified Eagle's medium supplemented with 10 % heat-inactivated fetal calf serum and 0.4% glutamine at 37" C in a 5 % COB-95 % air environment.The RPMI 8226 cells have the following types of biological properties: HLA BI 3 antigen (Associated Biomedic Systems); produce A light chains (Staneck et al., 1974;Moore and Kitamura, 1968; Matsuoka et al., 1967)