Two B-cell lines, a hairy cell leukemia (HCL) line (ZK-H) and a normal lymphoblastoid line (ZK-N), were established in long-term culture from Peripheral blood of the same patient with HCL. An anti-HCL serum was prepared by immunizing a rabbit with ZK-H cells, rendered HCL-specific by absorption with ZK-N cells and tested in indirect membrane immunofluorescence. The absorbed antiserum reacted with 3 of 3 HCL lines but not with another 21 hemic cell lines, including 4 non-T, non-B cell lines, 6 T-cell lines, 9 B-cell lines and 2 myeloid cell lines. When tested against fresh normal and leukemic cells, positive reactions were observed in 5 of 5 HCL cases but not in 6 normal persons or in another 26 cases of various leukemias, except for a low percentage positive reaction in 2 of 6 cases of CLL. These results suggest that HCL cells possess a specific HCL-associated antigen which is shared by certain CLL cells but which is distinct from a B cell antigen. The method of production of hetero-antiserum used in the present experiment appears to obviate problems related to histocompatibility antigeris and would be useful for the immunodiagnosis of leukemias.