We have examined a prediction made from the ribosome feedback regulation model of ribosomal RNA and transfer RNA synthesis in Escherichia coli. This model proposes that non-translating "free" ribosomes act directly or indirectly as negative feedback inhibitors to regulate the transcription of rRNA and tRNA operons. One prediction of this model is that preferential inhibition of the assembly of ribosomes (without inhibiting macromolecular synthesis) should lead to a deficiency of free ribosomes which should, in turn, cause a stimulation of rRNA (and tRNA) synthesis. We have examined this prediction in vivo by causing the preferential inhibition of synthesis of certain ribosomal proteins by an overproduction of the translational repressor ribosomal protein S4. In agreement with our model, we have observed a preferential stimulation of ribosomal protein messenger RNA and rRNA synthesis under these conditions. These results suggest that ribosomes in the cellular pool, rather than incomplete ribosomal particles or free rRNA, are monitored by cells to regulate the rate of rRNA synthesis, and give further support to this proposed model.