We recently established an Epstein-Barr virus (EBV)-positive γδ T-cell line from a nasal T/natural killer (NK)-cell lymphoma (Nagata H, Konno A, Kimura N, Zhang Y, Kimura M, Demachi A, Sekine T, Yamamoto K, Shimizu N: Characterization of novel natural killer (NK)-cell and γδ T-cell lines established from primary lesions of nasal T/NK-cell lymphomas associated with the Epstein-Barr virus. Blood 2001, 97:708–713). Subsequently, we established two novel EBV-positive γδ T-cell lines from the peripheral blood of patients with chronic active EBV infection. Analysis of the terminal repeat of EBV showed that the three cell lines consisted of monoclonal populations, and flow cytometry showed that they had a common phenotype of γδ T cells: CD3 + CD4 − CD8 − CD16 − CD19 − CD56 + CD57 − HLA-DR + T-cell receptor (TCR) αβ − TCR γδ +. Analysis for the expression of TCR by flow cytometry showed that all three cell lines were Vγ9 +/Vδ2 +, but negative for VγI, Vδ1, or Vδ3 TCR. Southern blot analysis for TCR genes showed that the three cell lines had a common rearrangement of Vγ9-JγP and Jδ3 genes. Polymerase chain reaction and sequence analysis of the junction between Vδ and Jδ genes revealed that the Jδ3 genes were rearranged with the Vδ2 genes. In contrast, none of the EBV-negative γδ T-cell lines, Molt-14, Peer, or Loucy, which were analyzed for controls, had Vγ9 or Vδ2 TCR, or a rearrangement of Jδ3 genes. These results indicated that Vγ9-JγP/Vδ2-Jδ3 + γδ T cells were preferentially affected by EBV and expanded in patients with nasal γδ T-cell lymphoma and chronic active EBV infection. Jδ3 + γδ T cells are known to be a very minor population in γδ T cells of peripheral blood, whereas Vγ9-JγP/Vδ2-Jδ1 + cells are the major population. The close association of EBV with this particular γδ T-cell population may provide a key to the etiology of EBV-positive lymphoproliferative diseases.