The role of the thymus in induction of leukemia was studied in vitro. Curltivation of normal thymus cells on thymus epithelial reticulum cell monolayers that had been grown from radiation leukemia virus-induced leukemic thymuses rendered the thymocytes leukemic. C57BL/6 thymocytes were cultivated for 3 days on leukemic thymus reticulum monolayers, and 106 thymocytes were injected i.p. into young adult C57BL/6 mice. After 3 to 4 weeks all mice died of disseminated lymphatic leukemia. Mice given thymocytes that had been cultivated on thymus epithelial reticulum monolayers from normal mice did not develop lymphomas. The leukemic thymus epithelial reticulum cells were shown to produce thymotropic as well as ecotropic and xenotropic radiation leukemia virus. (Thymotropic virus has affinity for thymus lymphocytes but noes not infect fibroblasts.) The cells were brightly positive for murine leukemia virus group-specific antigen in immunofluorescence tests. Leukemic thymus epithelial reticulum cells produced ample infectious exotropic virus in the culture supernatant, although the cells were negative in the XC syncytia test. Upon infection of mouse fibroblasts with ecotropic virus produced by the leukemic reticulum cells, XC syncytia were readily obtained. Thymocytes that were cultivated on leukemic thymus reticulum cells became positive for murine leukemia virus group-specific antigen and produced syncytia in the XC test. Thus, in vitro lymphomagenesis of the thymocytes that were cultured on leukemic thymus reticulum cells was associated with their infection with thymotropic and ecotropic radiation leukemia virus.