A key feature of transcriptional attenuation in some amino acid biosynthetic operons is a transcriptional pause that occurs immediately after synthesis of the first leader transcript secondary structure. Both RNA secondary structure and downstream DNA sequence are important for pausing at these sites; however, the precise RNA structures involved and the relative contribution of other RNA and DNA bases to pausing are unknown. We studied the effects of base substitutions upstream from the his leader pause site (immediately prior to addition of G103) to determine how nucleic acid sequences and RNA structure contribute to pausing. By testing compensatory base substitutions, we found that pausing depended in part on an RNA secondary structure containing a five base-pair stem and eight nucleotide loop, which we call the his pause RNA hairpin. The his pause hairpin forms 11 nucleotides upstream from the paused transcript 3' end and thus corresponds to only the upper portion of the larger his A:B leader transcript secondary structure. Some base substitutions in the ten nucleotides between the pause hairpin and the 3' end of the transcript increased pausing, whereas others decreased pausing. However, compensatory substitutions that restored pairing of these bases in the lower portion of the A:B secondary structure did not alter these effects. Changing the 3'-terminal nucleotide of the transcript (U102) altered both the position and strength of pausing. Thus, in addition to the downstream DNA sequence, three distinct segments of nucleic acid upstream from the nucleotide-addition site in the transcription complex contribute to pausing in different ways: the pause RNA hairpin, the 3'-proximal region of transcript or DNA template, and the 3'-terminal nucleotide. We suggest that electrostatic interaction between the pause hairpin and RNA polymerase, rather than disruption of an RNA:DNA heteroduplex, delays elongation at the his leader pause site.