A specific interaction between the alpha subunit of RNA polymerase and an A+T-rich upstream sequence (UP element) stimulates transcription at some promoters in Escherichia coli. We found that RNA polymerase formed a heparin-resistant nonproductive initiation complex at the malT promoter which has an A+T-rich upstream sequence that begins 9 bp upstream of the -35 region. Substitution of other sequences for the A+T-rich sequence eliminated both the formation of heparin-resistant complexes and alpha binding to the malT promoter. A 5-bp deletion between the A+T-rich sequence and the -35 region increased promoter activity. The UP element derived from the rrnB P1 promoter stimulated transcription of the malT core promoter when placed 4 bp upstream from the malT -35 region, but insertion of an additional 4 bp between the rrnB P1 UP element and the -35 element eliminated transcription activity without eliminating heparin-resistant complex formation. Similar UP element effects were observed in hybrids with the lac core promoter, even though the region around the transcription start site was melted in both productive and nonproductive complexes. We conclude that UP elements can mediate the formation of both productive and nonproductive open complexes, depending on their location with respect to the core promoter.