A RNA-DNA hybridization assay for ribonucleoside diphosphate reductase (RDP reductase) mRNA was used to determine whether control of RDP reductase synthesis in Escherichia coli is at the level of RNA transcription. The correlation observed between the level of RDP reductase enzymatic activity and the rate of RDP reductase mRNA synthesis suggested that the control is at the level of RNA transcription. No increase in RDP reductase enzymatic activity or RDP reductase mRNA was observed during the first 15 min after removal of thymine from a thymine-requiring culture. Thereafter, the rate of RDP reductase mRNA synthesis increased linearly for approximately 75 min before beginning to level off. The addition of thymine to a culture starved for thymine resulted in a decreasing rate of RDP reductase mRNA synthesis. However, 30 min of growth in the presence of thymine was required before the rate of RDP reductase mRNA synthesis dropped to the rate observed in an exponentially growing culture. Inhibition of DNA synthesis caused by shifting a culture of a polC mutant or a dnaB mutant to nonpermissive growth conditions resulted in an increase in the rate of RDP reductase mRNA synthesis similar to that observed for a thymine-starved culture.