Initiation of transcription is an important target for regulation of gene expression. In bacteria, the formation of a transcription-competent complex between RNA polymerase and a promoter involves DNA strand separation over a stretch of about 14 base pairs. Aromatic and basic residues in conserved region 2.3 of Escherichia coli sigma(70) had been found to participate in this process, but it is still unclear which amino acid residues initiate it. Here we report an essential role for threonine (T) at position 429 of sigma(70): its substitution by alanine (T429A) results in the largest decrease in open complex formation yet observed for any single substitution in region 2.3. Promoter recognition itself is not affected by T429A substitution, thus providing evidence for a role of T429 in the strand-separation step. Our data are consistent with a model where the T429 would act as a competitor for the hydrogen bonding that stabilizes the highly conserved -11A-T base pairs of the promoter DNA, thus facilitating initiation of strand separation at this particular position in the -10 region. This model suggests an active role for RNA polymerase in disrupting the -11 base pair, rather than just capturing the -11A subsequent to spontaneous unpairing.