A major membrane glycoprotein with mol wt of approximately 54,000 has been isolated from membrane preparations of B-type lymphoid cell lines. Antiserum prepared against the isolated material specifically precipitated this glycoprotein from membranes labeled by surface radioiodination or by metabolic labeling. This antiserum was shown by complement-mediated cytotoxicity assay, membrane immunofluorescent staining, and by quantitative absorption analysis to react preferentially with certain B-lymphoblastoid cell lines, with a minor population of peripheral blood B lymphocytes, and a major population of tonsillar B lymphocytes. Certain B-cell leukemias also expressed the antigen, whereas others did not. Considerable variability was observed among positive B cells in the intensity of fluorescent staining even among the leukemic cells from a single individual. Although T cells, including T cells, were negative by direct immunofluorescent and cytotoxicity assay, evidence for low levels of the antigen on the cells of T cell lines was obtained. The whole specific antiserum and its F(ab')2 fragments stimulated B lymphocytes to proliferate. This proliferation did not produce differentiation to plasma cells and was T-cell independent. The monovalent Fab fragments had no effect. None of these preparations timulated T cells. The possibility that this antigen, termed gp54, may play some role in B-cell activation is discussed.