Viral RNA present in the inducible LPT clone 1A of polyoma virus-transformed rat cells was characterized before and after mitomycin C induction by hybridization with 32P-labeled separated E and L strands of polyoma viral DNA restriction endonuclease fragments. In clone 1A cells maintained under normal growth conditions, the cytoplasm contained a transcript of the E-strand DNA from the "early" region similar to that previously identified in lytically infected cells, as well as minor quantities of RNA complementary to less than one-half of the L- and the E-strand DNA from the "late" region. Nuclei of normally growing cells contained the same species found in the cytoplasm, as well as an additional abundant RNA complementary to one-half of the L-strand DNA of the late region. No significant changes occurred in the cytoplasmic viral RNA after mitomycin C treatment before the onset of viral DNA replication, but the concentration of the nuclear L-strand DNA transcript diminished. After the onset of viral DNA replication after mitomycin C treatment, transcripts of virtually the entire L-strand DNA were found in the nuclei, and a 10-fold increase was observed in the abundance of RNA transcribed from the E strand of the early region. In the cytoplasm, the abundance of the early RNA increased about 25-fold and late RNA complementary to the L-strand DNA of the late region was found in a similar quantity. The synthesis of both the early and the late RNA species was inhibited if viral DNA replication was blocked with 5-fluorodeoxyuridine. We conclude that the induction of viral DNA replication in LPT cells is not determined at the level of mRNA synthesis.