Directly growing Epstein-Barr virus (EBV)-carrying cell lines were established from a chronic lymphocytic leukemia (CLL) patient (PG) on repeated occasions. The lines carried the same ring chromosome 15 as the leukemia cells in vivo and were similarly trisomic for chromosome 12. They all showed the same JH rearrangement, indicating that they had arisen from the same B-cell progenitor. They also had the same single EBV-terminal repeat (TR), indicating that they had been generated by a single EBV infection event. It may be surmised that a single CLL cell had been infected by EBV in vivo and established itself subsequently as a subclone within the CLL population. This subpopulation persists in vivo but does not appear to expand with time. After explantation, it transforms into lymphoblastoid cells and proliferates selectively as immortalized lines. The leukemia-representative CLL lines were phenotypically indistinguishable from the B95-8 virus-transformed normal diploid cells of the patient, established in parallel by in vitro infection. They grew as typical LCL clusters and expressed the same B-cell activation markers. The methylation status of EBV-DNA was different in the CLL lines and the B95-8-virus-transformed LCLs. When Hpall- and Mspl- digested DNA was probed with BamHI C, E, H and W fragments, the CLL lines showed a mixture of methylated and unmethylated restriction fragments as in certain EBV-carrying Burkitt lymphoma (BL) lines. In contrast, the EBV-DNA of B95-8 virus-transformed normal diploid cells was completely unmethylated, as in other LCLs.