Previous studies of the structure and regulation of the deo operon in Escherichia coli have localized an internal regulated promoter, called deoP3, in front of the two distal genes in the operon. We report here the nucleotide sequence of the distal portion of the deoA, the deoA-deoB intercistronic region and the first part of the deoB gene, and show that deoP3 overlaps the distal segment of the deoA gene. The location of the internal promoter and the transcriptional start site were determined by means of 1) sequence homology to the consensus promoter sequence of E. coli, 2) high resolution S1 nuclease mapping of in vivo transcripts and 3) in vivo regulation of beta-galactosidase from low as well as high copy number P31acZ protein fusion vectors.