The complete nucleotide sequence of the 23S RNA gene from the rrnB operon of *Escherichia coli* has been determined. The entire gene, spanning 2904 nucleotides, was sequenced using both strands and confirmed by overlapping restriction fragments. The sequence reveals the presence of kethoxal-reactive sites between positions 2300 and 3500, indicating the subunit interface. The 3' end of the 23S rRNA gene is supported by studies showing homology with chloroplast and mitochondrial rRNA. The sequence complements previous findings on 5S and 16S rRNA and completes the primary structural analysis of E. coli rRNAs. The sequence also provides insights into the structure and function of the ribosome, including potential roles in peptidyltransferase activity.The complete nucleotide sequence of the 23S RNA gene from the rrnB operon of *Escherichia coli* has been determined. The entire gene, spanning 2904 nucleotides, was sequenced using both strands and confirmed by overlapping restriction fragments. The sequence reveals the presence of kethoxal-reactive sites between positions 2300 and 3500, indicating the subunit interface. The 3' end of the 23S rRNA gene is supported by studies showing homology with chloroplast and mitochondrial rRNA. The sequence complements previous findings on 5S and 16S rRNA and completes the primary structural analysis of E. coli rRNAs. The sequence also provides insights into the structure and function of the ribosome, including potential roles in peptidyltransferase activity.