Spleen cells taken from W/Fu rats 4 to 6 weeks after immunization with the syngeneic Gross virus-induced lymphoma, (C58NT)D cells, at a time when they lack detectable activity in a short-term 51Cr release assay, were previously shown to retain the capacity to generate cytotoxic activity upon reexposure to mitomycin C-treated lymphoma (C58NT)D cells in vitro. In the studies presented here, we evaluated whether in vitro sensitization of immune lymphoid cells before systemic transfer to a nonimmune recipient allows for more effective transfer of tumor immunity. The results show that the passive transfer of immune spleen cells after in vitro cocultivation with mitomycin-treated (C58NT)D cells allows for inhibition of growth of a subcutaneous inoculum of lymphoma cells. In contrast, spleen cells obtained 4 to 6 weeks after primary sensitization or after secondary in vivo sensitization did not effectively confer anti-tumor immunity. As few as 5 x 107 in vitro sensitized cells permitted complete inhibition of 106 (C58NT)D cells and also allowed for inhibition of the growth of 107 (C58NT)D-F cells, which was lethal to control animals. Immune cells sensitized with syngeneic thymocytes or normal spleen cells sensitized with (C58NT)D cells in vitro did not confer in vivo anti-tumor immunity. After systemic transfer of in vitro sensitized cells, delayed hypersensitivity occurred at the site of tumor inoculation and tumor growth was suppressed. Specificity of the passive immunity was shown by the failure to inhibit growth of a polyoma virus-induced sarcoma in rats which inhibited growth of the Gross virus-induced lymphoma cells. In vitro sensitized cells were more effective in the transfer of anti-tumor protection after 5 days, as compared to 2 days, of cocultivation with tumor. Results show that in vitro sensitized cells can effectively transfer systemic tumor immunity.