We have analyzed the effect of gamma irradiation on the induction of morphological transformation of cloned rat embryo fibroblast (CREF) cells by the host-range cold-sensitive type 5 adenovirus mutant, H5hr1. Treatment of CREF cells with 1-6 Gy of gamma irradiation immediately prior to viral infection resulted in dose-dependent decrease in cell survival and concomitant increase in viral transformation frequency. Exposure of CREF cells to 1-6 Gy of gamma radiation alone resulted in a similar dose-dependent inhibition in cell survival but without any subsequent morphological transformation. The effect of gamma irradiation on viral transformation was greatest when cells were irradiated directly before viral infection. The reduction in the enhancement of transformation was both dose and time dependent. The ability of gamma irradiation to enhance viral transformation was substantially reduced if CREF cells were treated with inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Employing a single-cell colony transfer assay and in situ hybridization with a 32P-labeled Ad5 DNA probe, we found that gamma irradiation of CREF cells prior to infection with H5hr1 resulted, 10 and 17 d after infection and replating, in an increase in the percentage of surviving CREF colonies that contain Ad5 DNA. Analysis of viral DNA integration by DNA-filter hybridization (Southern blot analysis) in H5hr1-transformed CREF clones isolated from untreated and gamma-irradiated cultures indicates that gamma irradiation caused increases in both the number of copies of Ad5 E1A DNA sequences integrated into cellular DNA and the number of unique Ad5 E1A DNA integration sites in transformed cells. These results indicate that gamma irradiation enhancement of adenovirus transformation was a consequence of radiation-induced cellular factors with finite life spans that are mediators of enhanced viral transformation. Potentially important components of the radiation enhancement process appear to involve an alteration in both the retention of free Ad5 DNA in surviving cells and an alteration in the profile of viral-DNA integration in gamma-irradiated cells.