5-iododeoxyuridine (IdUrd) is highly effective in inducing the production of endogenous viruses (e.g., RNA-containing murine leukemia virus) from a variety of cell lines that normally do not release such viruses. For the activation of murine leukemia virus by IdUrd, its incorporation into cellular DNA is necessary. We have explored the possibility that incorporated IdUrd qualitatively alters the transcription of cell DNA. Nucleic acid hybridization between radioactive mouse unique DNA and RNA from the highly activatable mouse AKR-2B cell line indicates that the normal extent of transcription in AKR-2B cells is considerably lower than that observed in other lines of mouse cells studied. Treatment of AKR-2B cells with IdUrd increases the extent of transcription of unique DNA by 60%, which corresponds to an induction of approximately 2.5 X 10(4) gene equivalents. Included among this new set of RNA's are sequences that are transcribed from the DNA genome of the endogenous AKR-type murine leukemia virus present in AKR-2B cells. IdUrd treatment also markedly increases the synthesis and/or the accumulation of those RNA transcripts which are normally expressed in untreated cells. These results suggest that IdUrd stimulates the overall transcription activity of AKR-2B cells. It is possible that IdUrd-induced activation of endogenous murine leukemia virus is a consequence of this stimulation.