A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with beta 2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radiodinated components from similarily treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.