A CTL line (CTLL-D4) mediating specific cytolytic activity against radiation-induced leukaemia RL male 1 has been established and maintained on a long-term basis without the addition of exogeneous TCGF. This line was originally selected by the limiting dilution of MLTC cells from RL male 1-immune (BALB/c X C57BL/6) F1-nu/+(CB6F1-nu/+) spleen cells (500 cells/well) in the presence of 5% rat TCGF, 2000 rads-irradiated normal CB6F1-nu/+ spleen cells as the feeder cells, and 10,000 rads-irradiated RL male 1 tumour cells as the stimulator. After expansion only with the feeder and tumour cells, CTLL-D4 shows highly specific cytotoxic activity against RL male 1 by in vitro CMC assay, since cells such as RL male 6, RL female 8, RL female 9, P815, MOPC-315 (H-2d), EL-4 (H-2b) and YAC (H-2a) are not killed. Microcytoxicity assay of this line has revealed that CTLL-D4 comprises three subsets of T lymphocytes (100% Thy-1.2+): 15-25% Lyt-1+23-, 60-75% Lyt-1+23+ and 10-15% Lyt-1-23+. The proliferation of this line seems to depend largely upon the syngeneic MLR-like responsiveness of the Lyt-1+23- subsets of CTLL-D4 to the Ia-positive cells in CB6F1-nu/+ splenic feeder cells, and has been restricted to the H-2d-haplotype of the feeder cells. In spite of the vigorous cell proliferation by coculturing with the feeder cells alone, the cytolytic activity of this line begins to decrease after some 7 days of culture in the absence of the stimulator RL male 1 cells which have no capacity to stimulate by themselves. Thus, by long-term culture of CTLL-D4 with the syngeneic feeder cells alone, a new non-cytolytic line (D4f) was established. Mechanisms enabling the long-term maintenance of CTL activity and subset composition have been discussed in terms of cellular cooperation between the subsets of this line.