Resting T cells can be activated by selected pairs of anti‐CD2 MoAb. Activation is dependent on the presence of accessory cells, which can be replaced by either anti‐CD28, or by the combination of IL‐1β and IL‐6. The present study was undertaken to investigate accessory signalling by B7‐1, the natural ligandof CD28, in this pathway of T cell activation. 3T6 mouse fibrobiasts were transfected with human B7‐1 and used as accessory cells in cultures of purified resting human T cells. In the presence of a stimulating pair of anti‐CD2 MoAb, T cell proliferation, production of cytokines (IL‐2, IL‐4, IL‐10, GM‐CSF, IFN‐α and TNF‐α), and generation of cytotoxic T lymphocytes were all supported by B7‐l(+) 3T6 cells but not by control 3T6 cells. Blocking studies with anti‐IL‐2 + anti‐IL‐2R MoAb revealed both IL‐2‐dependent and IL‐2‐independent CTL generation after B7‐1 ‐mediated costimulation. Moreover, a partial or complete resistance to inhibition with CsA was observed for IL‐2 production and CTL generation respectively in the presence of the costimulatory signal derived from B7‐1 ‐ CD28 interaction. Anti‐CD2 MoAb with B7‐1 costimulation could directly induce proliferation, IL‐2 production and generation of CTL activity in highly purified CD8+T cells without the heip of CD4+T cells. We conclude that CD28 ligation with the natural ligand B7‐1 provides a str