In the diagnosis of non-Hodgkin's lymphomas, the ready characterization of the neoplastic cell lineage by analysis of cell surface markers is of great importance. We present evidence for the existence of a human B-leukemia-associated antigen recognized by a complement-fixing monoclonal antibody (anti-Y 29/55). A hybridoma was produced by fusing mouse myeloma cells and splenocytes of a mouse immunized against lymphoid cells of a patient with B-cell chronic lymphocytic leukemia. Characterization of anti-Y 29/55-reactive normal and malignant leukocytes was demonstrated by cytolysis and indirect immunofluorescence. This revealed reactivity with an antigen on B-lymphoma cells (11 patients), on leukemic lymphocytes in B-cell chronic lymphocytic leukemia (13 patients), and on malignant cells in hairy-cell leukemia (two patients) but not on leukemic cells of T-cell acute lymphoblastic leukemia (one patient), on T-lymphoma cells (one patient), on cells of acute myeloblastic leukemia (four patients), or of chronic myeloid leukemia (four patients). No specific cytolysis occurred with B- and T-peripheral blood lymphocytes from (a) healthy donors (16 individuals), (b) patient with reactive lymphocytosis (one patient), (c) nonleukemic multiple myeloma (six patients), or (d) Hodgkin's disease (three patients). Surface immunoglobulin-positive, sheep RBC-negative lymphocytes isolated from human spleen (three individuals), tonsils (seven individuals), and lymph nodes (one individual), however, were recognized. It is concluded that leukemic B-cells carry a marker characteristic of nonrecirculating sessile B-lymphocytes.