The primary activation pathway of T cells is via the T‐cell receptor (TCR)/CD3 complex, which is functionally interrelated with various accessory molecules. We examined the contribution of the lymphocyte‐function‐associated antigenI/intercellular adhesion molecule I (LFA‐I/ICAM‐I) interaction to CD3/TCR‐mediated lysis by cytotoxic T lymphocytes (CTL). We used ICAM‐I‐or+ tumor cell lines as target cells and anti‐CD3‐ or anti‐LFA‐I containing hetero‐cross‐linked monoclonal antibody (MAb) to bridge CTL and target cells and simultaneously to activate CTL. The ICAM‐I‐ melanoma‐derived cell line lgR39 was relatively resistant to CD3‐mediated lysis by both TCRαβ+ and TCRγdL+ CTL, when compared with ICAM‐I+ cell lines. Induction of ICAM‐I on the membrane of lgR39 cells by tumor necrosis factor (TNF) rendered these cells more susceptible to CD3‐mediated lysis. Anti‐ICAM‐I MAb inhibited this TNF‐enhanced susceptibility to lysis, directly demonstrating that the induction of ICAM‐I was critical in the TNF‐induced increase in susceptibility to lysis of lgR39 cells. CTL formed less efficient conjugates with the ICAM‐I‐ cells as compared to ICAM‐I+ cells. Both spontaneous and CD3‐induced conjugate formation as well as CD3‐mediated lysis of ICAM‐I‐ tumor cells by CTL were enhanced by the addition of anti‐LFA‐I containing heterocross‐linked MAb, thereby mimicking the LFA‐I/ICAM‐I interaction between CTL and target cells. Soluble anti‐CD18 MAb inhibited CD3‐mediated lysis of ICAM‐I‐ target cells by CTL without affecting their conjugate formation. Anti‐LFA‐I MAb added after conjugate formation still inhibited lysis of both ICAM‐I+or‐ tumor cells. Taken together, these findings suggest that the LFA‐I/ICAM‐I interaction co‐activates CD3/TCR‐mediated lysis by CTL through both an enhanced CTL‐target cell binding and the delivery of post‐conjugate costimulatory signals.