Permeabilized cells of Escherichia coli were used to examine the effect on RNA synthesis of guanosine 5'-diphosphate-3'-monophosphate (ppGp). Electrophoretic and hybridization analysis of bulk RNA demonstrated that ppGp selectively reduced the accumulation of both ribosomal and transfer RNAs. The experiments further suggested that the observed reduction in stable RNA accumulation resulted from a reduction in stable RNA chain initiation rather than an effect on elongation, processing, or maturation of stable RNA transcripts. In contrast, the expression of the lac Z gene was not affected by ppGp. These results suggest that ppG, a nucleotide produced in E. coli during the stringent response, could play a direct role in the stringent regulation of stable RNA synthesis.