A temperature-sensitive mutant of E. coli, rne (RNAase E-), accumulates a number of relatively small RNA molecules at the nonpermissive temperature. One of these molecules, 9S, contains 5S rRNA sequences. The 9S RNA can be processed in vitro to 5S (p5) and a 4S-sized molecule. The 9S and the p5 and 4S RNAs processed from it were all characterized by RNAase T1 fingerprinting and pancreatic RNAase redigestion analysis. Unique oligonucleotides found in the p5 and the 4S RNA are present as unique oligonucleotides in the 9S RNA, suggesting that each 9S molecule contains one 5S and one 4S RNA moiety. Since the 9S RNA contains about 420 nucleotides, and intervening sequences are not known to exist in E. coli, the 5S an 4S RNAs cannot be more than 200 nucleotides apart in the genome. E. coli contains at least three different sequence variants of 5S rRNA, all of which can be identified in the 9S transcript, indicating that 9S RNA is transcribed from most, if not all, of the active rRNA genes, and that RNAase E processes transcripts derived from all these rRNA genes. No modified bases were found in the 4S RNA, nor was its sequence compatible with the known sequences of tRNAs found near the 5S rRNA. We therefore conclude that the 9S RNA doses not contain tRNA, and that the tRNA molecules located near the 5S rRNA are distal to it.