One approach to adoptive cancer immunotherapy is based on the use of bispecific monoclonal antibodies (mAb) capable to redirect ex vivo generated cytolytic T lymphocytes (CTL) onto tumour cells. The efficiency of the CD28 T-cell activation pathway to induce CD3-dependent cytolytic activity was investigated while avoiding modulation of the TCR/CD3 complex needed for targeting by bispecific mAb. When used e.g. in conjunction with anti-CD2 antibodies or diacylglycerol derivatives, the in vitro stimulation of T cells with anti-CD28 mAb resulted within 36 h in high levels of CD3-dependent cytolysis (tested on a FcR+ target in the presence of anti-CD3 mAb) and sustained lymphokine production, such as TNF alpha, IFN gamma and IL-2, which may affect tumour growth when delivered locally by the transferred T cells. Rapid activation may reduce costly in vitro procedures, preserve homing capacities of retransfused T cells, and thus facilitate implementation of clinical trials based on the use of bispecific antibodies.