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A novel cell line, FL-18, was established from the pleural effusion of a patient with follicular small cleaved cell lymphoma. At the same time, an Epstein-Barr virus (EBV) nuclear antigen (EBNA)-positive cell line, FL-18-EB, was established from the EBV-infected culture of the same pleural effusion cells. Both cell lines had the same monoclonal surface immunoglobulin (IgG kappa), and they had the same karyotype as that of the fresh pleural effusion cells in which a reciprocal translocation between the long arm of chromosomes 14 and 18 [t(14;18)(q32;q21)] was detected. Gene rearrangement analysis of immunoglobulin heavy-chain gene (JH) and kappa light-chain gene (J kappa) showed the same rearranged configuration in the two cell lines; however, some morphological and phenotypic differences were found. The FL-18-EB cells, which were morphologically similar to common EBNA- positive lymphoblastoid cell lines of normal B cell origin at the initial phase of culture, were larger than the FL-18 cells and contained multinucleated giant cells. The FL-18 cells lacked cytoplasmic immunoglobulin and were positive for common acute lymphoblastic leukemia antigen (CALLA), whereas the FL-18-EB cells had cytoplasmic immunoglobulin and were negative for CALLA. Thus, the phenotype of FL-18-EB seems to be a result of a shift by EBV infection to a more mature stage in the B cell differentiation pathway than that of FL-18. The paired availability of EBV-free and EBV-infected cell lines of a neoplastic clone is unique and valuable in considering EBV infectibility of neoplastic B cells and resultant phenotypic changes.