A DNA sequence rich in (A plus T), located upstream of the minus 10, minus 35 region of the Escherichia coli ribosomal RNA promoter rrnB P1 and called the UP element, stimulates transcription by a factor of 30 in vivo, as well as in vitro in the absence of protein factors other than RNA polymerase (RNAP).When fused to other promoters, such as IacUV5, the UP element also stimulates transcription, indicating that it is a separable promoter module. Mutations in the carboxyl-terminal region of the alpha subunit of RNAP prevent stimulation of these promoters by the UP element although the mutant enzymes are effective in transcribing the ``core'' promoters (those lacking the UP element). Protection of UP element DNA by the mutant RNAPs is severely reduced in footprinting experiments, suggesting that the selective decrease in transcription might result from defective interactions between alpha and the UP element. Purified alpha binds specifically to the UP element, confirming that alpha acts directly in promoter recognition. Transcription of three other promoters was also reduced by the COOH-terminal alpha mutations. These results suggest that UP elements comprise a third promoter recognition region (in addition to the minus 10, minus 35 recognition hexamers, which interact with the sigma subunit) and may account for the presence of (A plus T)-rich DNA upstream of many prokaryotic promoters. Since the same alpha mutations also block activation by some transcription factors, mechanisms of promoter stimulation by upstream DNA elements and positive control by certain transcription factors may be related.