The Thy-1 molecule on murine T lymphocytes has been suggested to play a role in cellular activation events leading to a variety of immunologic functions. We present evidence that this molecule may be involved in signals leading to the in vitro generation of cytotoxic T cells against several tumor cell lines used as stimulators in mixed tumor-lymphocyte culture. The presence of monoclonal antibody against a polymorphic determinant on the Thy-1 molecule markedly reduced the generation of cytotoxicity after three days of culture of murine splenocytes with stimulator tumor cells bearing low levels of Ia antigen. In contrast, no effect was seen when the stimulators were either allogeneic splenocytes, or a tumor cell line expressing large amounts of Ia. These results suggest that the Thy-1 molecule is critically involved in events leading to the generation of cytotoxic effectors under some, but not all conditions.