EBV-specific cytotoxic T cells (Tc) induced in vitro have continuously proliferated in vitro for over 9 months. The long-term maintenance of the Tc growth was dependent on periodic supplementation of both irrediated EBV-transformed autologous lymphoblastoid cell line (LCL) cels and conditioned medium. The latter was derived from supernatants of human spleen-cell cultures stimulated with phytohemagglutinin. The cultured Tc maintained significant cytotoxic activity to autologous LCL cells but not to EBV-unrelated target cells, including K-562, B-, T-, null-cell lines and mitogen-stimulated antologous peripheral blood lymphocytes. Thus, established EBV-specific Tc lines from five different individuals always exhibited highly significant cytotoxicity against exhibited highly significant cytotoxicity against autologous LCL cells but not always against allogeneic LCL cells. Furthermore, restriction of the Tc to the autologous LCL was more pronounced after long-term culture than it was initially. This suggests that certain clones of Tc which arignificant cytotoxicity against exhibited highly significant cytotoxicity against autologous LCL cells but not always against allogeneic LCL cells. Furthermore, restriction of the Tc to the autologous LCL was more pronounced after long-term culture than it was initially. This suggests that certain clones of Tc which arignificant cytotoxicity against exhibited highly significant cytotoxicity against autologous LCL cells but not always against allogeneic LCL cells. Furthermore, restriction of the Tc to the autologous LCL was more pronounced after long-term culture than it was initially. This suggests that certain clones of Tc which are probably restricted to HLA are selectively established during long-term cultivation.