The promoter of the Escherichia coli udp gene contains the poly-T (5'-TTTTT-3') motif in the transcription start region located at the distance of 3 nucleotides with respect to the Pribnow box. By means of site-directed mutagenesis, mutations in the +1, -1, and +3 positions of this region were isolated and their functional role in transcription initiation was determined. It was shown that in addition to the thymine nucleotide earlier identified at position 4 (with respect to the 5' end of the poly-T motif), the third thymine nucleotide may serve as an efficient transcription site. The functional significance of the presence of the poly-T motif in the transcription initiation region is discussed. Analysis of the isolated mutant promoters revealed the ability of the guanine nucleotide at the +3 position to stabilize the formation of a productive transcription complex and to serve as an additional transcription start.