Transcription initiation has been shown to occur in vitro at several sites within a cloned Caulobacter crescentus ribosomal RNA gene cluster that lacks the major promoter region 5' to the 16 S rRNA gene. The predominant transcription start site in vitro was located near the 3' end of the 16 S rRNA gene. Transcription initiation from this region was also detected in vivo, when the cloned rRNA gene cluster was present on a multi-copy plasmid. The transcription start sites in vitro and in vivo were shown to be identical by S1 nuclease mapping and were found to be located approximately 300 nucleotides upstream from the 3' end of the 16 S rRNA gene. The transcript synthesized in vitro was shown to be cleaved by C. crescentus RNase III and to release the transfer RNA genes from the downstream 16 S/23 S intergenic spacer region. Analysis of the nucleotide sequence near the internal 16 S rRNA transcription start site revealed the presence of a consensus promoter sequence followed by the beginning of an open reading frame approximately 90 nucleotides downstream. Examination of the 16 S rRNA genes from other bacterial species and chloroplasts and 18 S rRNA genes from Xenopus and yeast revealed that the nucleotide sequence of this internal 16 S rRNA promoter region was highly conserved. Although the length of these 16 S and 18 S rRNA genes is slightly variable, the distance of the conserved promoter sequence from the 3' end of these genes has been conserved.