We have developed a system to induce oncornavirus-specific secondary cytotoxic response in vitro. When Moloney strain of murine sarcoma virus-immune spleen cells were cultivated with purified infectious Moloney murine leukemia virus (M-MuLV) or with supernates of tissue culture cells containing infectious virus, a virus-specific secondary cytotoxic response directed against type-specific determinant(s) of M-MuLV was generated in vitro, as determined by a 4-h 51Cr-release assay. The effector cells were susceptible to the treatment with anti-Thyl.2 plus complement, but were unrelated to natural killer cells (NK), because they could not lyse some target cells specific for M-MuLV in both the induction phase and the interaction between effector cells and target cells. Furthermore, a product of the env gene of M-MuLV, perhaps gp70, appeared to be responsible for this response, because viruses with recombinations in the env gene between ecotropic M-MuLV and a xenotropic virus failed to induce a response. When infectious M-MuLV was exposed to UV-light at different doses, the ability of UV-treated M-MuLV to induce a secondary cytotoxic response decreased in parallel with infectivity, indicating that infectivity was necessary for the induction of this response.