A hairy-cell leukemia (HCL) line, BNBH-I, was established from the peripheral blood of a 40-year-old male patient with HCL in a relatively stable clinical phase after splenectomy. The cells have since been growing continuously for more than 2 years. Their cell surface immunoglobulin (sIg) was identical with that found on the surface of freshly isolated leukemic cells, consisting of IgG-kappa. The BNBH-I cells were more mature than the original hairy cells in their degree of B-cell differentiation, as reflected by a decrease in sIg expression together with the appearance of some cytoplasmic Ig (cIg)+ cells, loss of EA gamma-rosette formation and reactivity with monoclonal antibody (MAb) FMC7, and an increase in the proportion of MAb PCA-I+ cells. The BNBH-I cells possessed the antigen recognized by Leu-M5, a highly specific MAb for HCL. Epstein-Barr virus nuclear antigen (EBNA) was present. Both the freshly isolated leukemic cells and the cell line had the 14q+ involving q32 chromosomal abnormality, and their Ig gene rearrangements were also identical. Following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA), both the freshly isolated leukemic cells and the BNBH-I cells adhered to culture dishes and extended long, thin processes, a phenomenon characteristic of HCL. These results indicate that the BNBH-I line was derived from the leukemic hairy cells.