The Escherichia coli promoter p_BAD, under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at p_BAD overlaps the RNA polymerase 35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the 35 region or whether both AraC and sigma make important contacts with the DNA in the 35 region. If sigma does not contact DNA near the 35 region, p_BAD activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the p_BAD promoter and in a second promoter where the AraC binding site overlaps the 35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the 35 region. Since sigma and AraC are thus closely positioned at p_BAD, it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.