Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate- resistant acid phosphatase (TRAP)-positive “hairy” lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S- HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation- preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.