Cytotoxic lymphocytes (CTL) to allogeneic and syngeneic murine leukemia cells were generated in vitro in "one way" mixed lymphocyte-tumor cell cultures carried out under a variety of conditions. In an attempt to define the optimal culture conditions for sensitization, the following parameters were analyzed: culture vessel, culture volume, responder: stimulator (R:S) cell ratio, lymphoid cell density, source of lymphoid cells, duration of culture, fetal calf serum (FCS) concentration, 2-mercaptoethanol (2-ME) concentration, concentration of amino acids, and buffering system. Of the many variables examined, of particular importance were the R:S cell ratio, the cell density, the FCS concentration, the presence of 2-ME, and the time of harvesting. These factors exerted various effects, both quantitatively and qualitatively, on the magnitude and kinetics of the responses induced in microcultures (5 X 10(6) lymphocytes) and macrocultures (25 X 10(6) lymphocytes). Moreover, optimal sensitization in syngeneic cultures required different conditions than those for allogeneic cultures. Cytotoxic activity was assessed in vitro by a quantitative 51Cr-release assay. The sensitized lymphocytes demonstrated the characteristics ofT lymphocytes and reacted specifically with the sensitizing leukemia cells.