Rabbit antibodies to bovine S-100 protein were tested by immunoperoxidase technics against fresh hairy cell leukemia (HCL) cells obtained from nine patients (peripheral blood in six and spleen in three), as well as lymphoblastoid cell lines derived from three patients with HCL. Peripheral mononuclear cells from three normal persons and two patients with chronic lymphocytic leukemia (CLL) and cells from two melanoma lines were used as controls. The melanoma cell lines, cell lines derived from patients with HCL, and fresh HCL cells displayed cytoplasmic and nuclear positivity after exposure to anti-S-100 protein sera. By contrast, normal peripheral blood lymphocytes and CLL cells were negative for S-100 protein. Additional studies were performed by immunoperoxidase technics on representative sections of formalin-fixed splenic tissues from eight patients who had splenectomies. The cause of splenectomy was HCL in three, traumatic rupture in one, CLL in one, Hodgkin's disease in one, and hypersplenism in two patients. Sections from all three HCL patients showed moderate to marked positivity with antisera to S-100 protein. These results strongly suggest the presence of S-100 protein in HCL cells.