The nucleotide sequence of a cloned gene for the RNA component of Escherichia coli ribonuclease P, M1 RNA, is presented. The sequence determined extends 320 nucleotides upstream of the 377-base-pair (bp) structural gene and includes three sequences homologous to the consensus E. coli promoter sequence. Two nucleotides found in the M1 RNA structural gene sequence were not found in a previously determined gene sequence of another M1 RNA clone [Reed, R. E., Baer, M. F., Guerrier-Takeda, C., Donis-Keller, H. & Altman, S. (1982) Cell 30, 627-636]. In vitro transcription of supercoiled plasmid DNA containing the M1 RNA gene resulted in a major transcript arising from the strong promoter nearest to the mature M1 RNA. RNAs encoded by the M1 RNA clone in vivo were examined by S1 nuclease mapping. The results indicated that in vivo transcripts originate from all three promoters preceding the M1 RNA gene. These transcripts are apparently processed in a multistep pathway to generate the 5' end of mature M1 RNA.