In previous studies we observed that resistance of murine SV40-transformed fibroblast cell lines to cytolysis by activated macrophages was frequently associated with cellular expression of the gp70 of an endogenous ecotropic murine leukemia virus (MuLV). The work described here was initiated to test directly for a causative relationship between MuLV expression and resistance to lysis by macrophages. Northern blot analysis revealed that macrophage-resistant cells contain full length retroviral RNA. A panel of mAb which distinguish among host-range classes of MuLV detected only a non-recombinant ecotropic gp70 in these cells. The ecotropic MuLV from two independently derived macrophage resistant cells were isolated by limiting dilution cloning on Mus dunii fibroblasts. These viruses were then used to infect macrophage-sensitive cell lines and the resultant MuLV-positive cells tested for sensitivity to macrophage cytolysis. The MuLV-infected lines remained highly sensitive to macrophage lysis despite their high levels of cell surface gp70 and release of infectious MuLV. Thus, although we cannot rule out the possibility that MuLV or a product thereof is necessary for development of macrophage resistance in transformed cells, expression of MuLV per se is not sufficient to create the resistant phenotype.