R1-20, a novel mAb reacting with a cell surface Ag on normal human lymphocytes and leukemic cell lines, was shown to induce homotypic cell aggregation in leukemic cell lines. This phenomenon was specific to mAb R1-20 because antibodies recognizing CD2, CD7, CD28, and HLA-ABC failed to exhibit homotypic cell aggregation. Induction of aggregation by mAb R1-20 occurred at 37 degrees C, but not at 4 degrees C and required cytoskeletal integrity. Sodium azide, a metabolic inhibitor, had no effect on the aggregation. Distinct from lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction in which divalent cations are essential elements, R1-20-mediated aggregation was not abolished with EDTA treatment. The R1-20 Ag was determined as a molecule of M(r) 100 to 110 kDa in immunoprecipitation and immunoblotting methods, under both reducing and nonreducing conditions. The molecular composition is quite different from that of any known integrin molecule. The R1-20 Ag was expressed on resting and activated T Lymphocytes as well as on normal B lymphocytes. Monocytes and granulocytes had no detectable R1-20 Ag. Among the leukemia-derived cell lines we used, mAb R1-20 reacted with 18 of 32 T cell lines, 2 of 20 B cell lines, 2 of 3 non-T-non-B cell lines, 2 of 7 myelomonocytic cell lines, and 2 of 3 nonlymphoid-nonmyeloid cell lines. All EBV-transformed B cell lines examined (10 cell lines) were R1-20+. The spectrum of reactivity among the cell lines tested was different from that of known antiadhesion antibodies tested. All these findings indicate that the Ag recognized by mAb R1-20 may represent a new type of cell adhesion molecule.