The single RNA operon of Mycoplasma pneumoniae was cloned into a lambda vector and subcloned into pBR322. This was carried out in order to enable the analysis of the transcription control regions of this operon. S1 nuclease mapping was used to locate the 5' ends of RNA transcripts synthesized from the operon. The 5' ends of the 23S, 16S, and a precursor RNA synthesized in vivo in M. pneumoniae were mapped on the DNA template. Preliminary in vitro transcription experiments using RNA polymerase of Escherichia coli led to the conclusion that E. coli recognizes one promoter in the 5' region of the M. pneumoniae rRNA operon. The startsite of the in vitro transcript seems to lie downstream from the 5' end of the M. pneumoniae precursor transcript. Preliminary sequencing of the 5' regions of the M. pneumoniae rRNA operon and of the M. capricolum rRNA B operon enabled their comparison to each other and to known sequences from other organisms.