The 5'-terminal sequences of Escherichia coli ribosomal RNA precursors (pre-rRNAs) synthesized in vivo were characterized by RNA oligonucleotide sequence analysis. The 60- to 170-nucleotide-long 5'-end-specific fragments were produced by RNase III treatment of 30S and 18S pre-rRNAs. Comparison of the RNA oligonucleotides of these fragments with known DNA sequences of the promoter regions of several ribosomal RNA operons allows us to determine the start points of transcription of each operon. We conclude that transcription of most (and perhaps all) rRNA operons is initiated in vivo at two tandem promoters, called P1 and P2, which have recently been identified by in vitro transcription studies of several groups. Depending on the transcription unit, the initiating nucleotide at P1 promoters is either ATP or GTP, whereas at P2 promoters it is either CTP or GTP.