DNA from virus-negative AKR mouse embryo cells is infectious for NIH 3T3 cells if the transfected cells are treated with 5-iododeoxyuridine (IdUrd) either before or after addition of the DNA. The virus isolated after transfection of the AKR DNA is an ecotropic murine leukemia virus (MuLV) Previous transfection studies have shown that retroviral DNA is infectious either as an unintegrated proviral DNA genome from acutely infected cells (1-4) or as an integrated proviral DNA genome from chronically infected cells (5-10). However, DNA from cells known to contain unexpressed but potentially infectious endogenous viral genomes is not infectious under the same transfection conditions (11). Thus, except for the demonstration that DNA from avian cells can complement the reverse transcriptase lesion in a temperature-sensitive mutant of Rous sarcoma virus (12), DNA from normal cells has not yet been shown to be infectious, despite genetic, biologic, and biochemical studies that indicate that endogenous retroviral genomes are present in cells from many species (13,14). The demonstration of complete retroviral expression from the DNA of normal cells would show directly that the cell DNA is potentially infectious. In addition, an infectivity assay for an endogenous viral genome would be useful in attempting to elucidate the cellular and viral mechanisms that restrict the expression of the viral genome in the endogenous state and permit its expression in the productively infected state.Although all inbred mice (Mus musculus) apparently contain endogenous retroviral genomes (15, 16) that are potentially infectious (17), strains vary markedly in their viral expression in vivo and in vitro (18)(19)(20)(21) Chemicals. IdUrd was purchased from Sigma and it was stored at 1 mg/ml in sterile phosphate-buffered saline at -20°C.DNA Isolation. Bulk cellular DNA was isolated as described (30) except that, prior to treatment with RNase, the ethanolprecipitated nucleic acid was wound on a rod instead of being concentrated by centrifugation. This modification was used because the centrifuged DNA was more difficult to redissolve than was the "spooled" DNA. This DNA had an average molecular weight of greater than 5 X 107. It was stored at 150-300 ,g/ml (20 A260 units/,ug DNA) at 4°C in 10 mM NaCl/10 mM Tris, pH 8.0 /0.5 mM EDTA. Stored DNA is still infectious after 1 year.Because it was critical that untreated AKR 2B cells not be producing virus at the time of DNA isolation, at least 2 X 106 AKR cells from the pool of cells used for each AKR DNA isolation were cocultivated with SC-1 cells, passaged at least twice, and found at that time to be negative for MuLV by the XC plaque test (31). IdUrd-treated AKR DNA was obtained after 24-hr treatment of AKR 2B cells with IdUrd at 40,g/ml. Incorporation of IdUrd into DNA was confirmed by CsCl density gradient centrifugation. Infectious center plating onto SC-1 cells Abbreviations: MuLV, murine leukemia virus; IdUrd, 5-iododeoxyuridine.