Spleen cells from BALB/c or CAF(1) mice released little or no detectable leukemia virus when cultured 2-7 days in vitro. In contrast, spleen cells of CAF(1) mice previously inoculated with parental BALB/c spleen cells released leukemia viruses in 10 of 11 cases studied. Cultures of a mixture of spleen cells from normal BALB/c and CAF(1) mice also contained leukemia viruses. Phytohemagglutinin induced the transformation of lymphocytes in cultures of CAF(1) or BALB/c spleen cells, but this transformation did not activate leukemia viruses. It is concluded that mixed lymphocyte cultures in vitro, just as graft-versus-host reactions in vivo, can activate leukemia viruses that are normally present in a repressed form. This activation is not solely a function of lymphocyte transformation. The activated mouse leukemia virus may subsequently account for the observed high incidence of neoplasia in graft-versus-host disease.