In order to assess the role of Epstein-Barr virus (EBV) in patients with hairy cell leukemia (HCL), we have sought to characterize 1) the ability of EBV to infect and transform hairy leukemic cells in vitro and 2) the phenotypes of cell lines putatively derived from those leukemic cells. Analysis of EBV-induced transformation and the kinetics of Epstein-Barr nuclear antigen (EBNA) induction in leukemic preparations indicated that most leukemic cells were not susceptible to EBV infection but that at least a small subpopulation of leukemic cells could be infected with EBV. Lymphoblastoid cells lines were established after exposure of peripheral blood or splenic cells from HCL patients to B95-8 or QIMR-WIL EBV. Splenic leukemic cell preparations were more sensitive targets for EBV transformation than were peripheral blood cell samples. The newly established cell lines, but not long-established B lines such as Raji, demonstrated high levels of synthesis of p35, (a protein complex expressed abundantly by cells of a subset of HCL patients) and high levels of tartrate-resistant acid phosphatase (an enzyme relatively diagnostic for HCL). Lymphoblastoid lines from one patient with HCL expressed lambda light chains and no kappa chains as did the patient's leukemic cells. Virus expression in these lines showed that HCL-derived lines had spontaneous early antigen (EA) and viral capsid antigen (VCA) expression. Transforming EBV could be rescued from HCL-derived cell lines but not from cord blood-derived lines.