Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs that recognize the invariant CD3 signaling complex is a controlled polyclonal activation of T cells that, ideally, is exquisitely dependent on the presence of target cells. Otherwise, overt production of inflammatory cytokines and secondary reactions may occur as side effects, as can be observed with constitutively T-cell activating monoclonal antibodies to CD3 or CD28, and with bispecific antibodies bearing Fc gamma portions. Here we analyzed 2 distinct bispecific single-chain antibody constructs of the BiTE class, called MT110 and MT103 (or MEDI-538), for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of the BiTE molecules were sufficient to stimulate a high percentage of peripheral human T cells to express cytokines and surface activation markers, enter into cell cycle, and induce redirected lysis of target cells. However, in the absence of target cells, the 2 BiTE molecules even at high concentrations did not detectably activate T cells. Our data show that T cell activation by monomeric forms of MT110 and MT103 is highly conditional in that it is strictly dependent on the presence of cells expressing the proper target antigen. BiTE molecules therefore qualify for a highly controlled polyclonal T-cell therapy of cancer.