A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16 S and 23 S genes of Escherichia coli ribosomal RNA operons. We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription. In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E. coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo. The rrnB mutant constructs contained an additional point mutation in each of the 16 S and 23 S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis. Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis. Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis. In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium. This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA. We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA.