The transcription initiation sites of E. coli rRNA operons were determined using various DNA fragments derived from transducing phage lambda metA20 carrying rrnE and from hybrid plasmid pLC19-3 carrying rrnA. In vitro transcription products were analyzed for their 5' end sequences and their oligonucleotide compositions. The results are in full agreement with the nuceotide sequences of the DNA templates described in an accompanying paper (de Boer, Gilbert and Nomura, 1979) and allow us to make the following conclusions. First, there are two transcription, start sites on each of the rRNA operons; they are 109 bp apart in the case of rrnE and 117 +/- 1 bp aprart in rrnA. Second, the first start site is 283 bp upstream from the m16S rRNA coding region in the case of rrnE, while is 291 bp upstream in rrnA. Initiation starts with ATP in both cases. Finally, the second start sites are 174 and 174 +/- 1 bp from the m16S rRNA genes in rrnE and rrnA, respectively. Initiation starts with CTP in both cases. We have also shown that in the present in vitro transcription system, guanosine tetraphosphate (ppGpp) inhibits the synthesis of full-sized RNAs from both start sites in each rRNA operon.