rRNA synthesis is the rate-limiting step in ribosome synthesis in Escherichia coli. Its regulation has been described in terms of a negative-feedback control loop in which rRNA promoter activity responds to the amount of translation. The feedback nature of this control system was demonstrated previously by artificially changing ribosome synthesis rates and observing responses of rRNA promoters. However, it has not been demonstrated previously that the initiating nucleoside triphosphate (iNTP) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp), the molecular effectors responsible for controlling rRNA promoters in response to changes in the nutritional environment, are responsible for altering rRNA promoter activities under these feedback conditions. Here, we show that most feedback situations result in changes in the concentrations of both the iNTP and ppGpp and that the directions of these changes are consistent with a role for these two small-molecule regulators in feedback control of rRNA synthesis. In contrast, we observed no change in the level of DNA supercoiling under the feedback conditions examined.