To define cell surface antigens associated with hairy cell leukemia (HCL), and to gain better insight into the origin of this disease, we developed monoclonal antibodies against spleen cells of a patient with this disease. Although none of these antibodies alone proved specific for the leukemic cells, two of them, designated alpha S-HCL 1 (alpha Leu-14) and alpha S-HCL 3 (alpha Leu-M5) were found to be valuable in the diagnosis of HCL when used in combination. alpha S-HCL 1 recognizes an antigen associated with greater than 95% of B cells in the peripheral blood. Biochemical analysis identified this antigen as a single polypeptide chain with a molecular weight of 150,000 daltons (150 kilodaltons). alpha S-HCL 1 expression on hairy cells is markedly increased when compared with normal B lymphocytes isolated from peripheral blood, tonsils, and spleens. alpha S-HCL 3 reacts with an antigen present on hairy cells but also on monocytes, macrophages, in a lower density on neutrophils, and a small percentage (less than 2%) of lymphocytes. The antigen recognized by alpha S-HCL 3 is composed of a non-covalently linked biomolecular complex of 90 and 150 kilodaltons. Since the HCL 3 antigen was not detectable on other lymphomas of either T or B cell type, the co-expression of S-HCL 1 and S-HCL 3 on hairy cells is a unique marker for this disease.