Permanent, non-virus-producing cell lines have been established from a mouse embryo carrying an endogenous, genetically transmitted Moloney murine leukemia virus (M-MuLV) genome. These cells carry the M-MuLV genome, as demonstrated by hybridization of cellular DNA to M-MuLV complementary DNA, but do not express it at the levels of virus production, accumulation of intracellular viral p30, or M-MuLV-specific RNA. Treatment with bromodeoxyuridine (50 microgram/ml for 24 h) resulted in induction of XC-positive NB-tropic virus, although only a small fraction of the cells released virus (less than 0.1% after 48 h). Immunofluorescent staining and flow microfluorometry indicated that a wave of p30 accumulation occurs in the induced cells, with a maximum at 24 to 48 h after the addition of bromodeoxyuridine. Furthermore, most, if not all, cells were induced to produce p30 protein. Similar kinetics were found for the accumulation of M-MuLV-specific RNA in the cytoplasm of induced cells. This rapid induction of virus expression in a majority of cells was dependent on the presence of the M-MuLV genome and probably represents primarily the expression of this endogenous virus since induction was not observed in cells similarly derived from a sibling embryo lacking the M-MuLV genome.