Monoclonal antibodies to murine lymphocyte differentiation antigens were generated by fusing the mouse myeloma cell line X63-Ag8.653 with spleen cells derived from Lewis rats hyperimmunized with lymphoid cells from nude (C57BL/6) mice. One of these antibodies--designated 3MB1--recognizes a previously undescribed cell surface antigen. This antigen is expressed on 62% of spleen cells, 9% of thymus cells, and 98% of peritoneal macrophages. Virtually all LPS- and Con A-induced lymphoblasts carry this newly found antigen. The 3MB1 determinant is limited in its expression to leukocytes. It is not found in other tissues such as brain, liver, kidney, or on erythrocytes. Biochemical analysis reveals that the 3MB1 target antigen consists of a single chain glycoprotein with an approximate m.w. of 90,000.