When P3HR-1 lymphoblastoid cells expressing Epstein-Barr virus (EBV)-related antigens at very low frequency were cocultivated with human umbilical cord blood lymphocytes and the cell-free mixed culture fluid was applied to fresh cord lymphocytes, cells morphologically distinct from normal lymphocytes became evident after one to two weeks' exposure. The abnormal cells became abundant after one month and were easily identified by B-cell markers and a variety of morphologic abnormalities. Such abnormal B-lymphocytes appeared to be negative for EBV-determined nuclear antigen (EBNA), but when the immunofluorescence-negative cells were exposed to pokeweed mitogen and 5-iododeoxyuridine, a striking EBNA induction occurred. The growth of these abnormal cells was limited and they could be maintained for no more than three months. The implications of these findings are discussed in relation to the biological activity of EBV.