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In Escherichia coli, primary rRNA transcripts must be processed by a complex process in which several ribonucleases are involved in order to generate mature 16S, 23S, and 5S rRNA molecules. While it is known that RNase G, a single-stranded RNA-specific endoribonuclease encoded by the rng gene, plays an active role in the maturation of the 5′-end of 16S rRNA, its involvement in the maturation of the 5′-end of 23S rRNA remains unclear. Here we show that E. coli cells deleted for the rng gene accumulate the 23S rRNA precursor containing an extra 77 nucleotides at its mature 5′-end. In vitro cleavage assays show that RNase G cleaves synthetic RNA containing a sequence encompassing the 5′-end to 77 nucleotides upstream of mature 23S rRNA at two sites present in single-stranded regions. Our results suggest the involvement of RNase G in the processing of the 5′-region of 23S rRNA precursors.