In Escherichia coli, pyrimidine-mediated regulation of upp expression occurs by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be elongated. The upp initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand). Initiation can occur at either the first or the second base in this sequence (designated G6 and AT, with numbering from the promoter -10 region). High intracellular UTP levels favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive UMP addition) within the 8-bp T · A tract in the initially transcribed region and are aborted. In contrast, low intracellular UTP levels favor initiation at position G6, which results in transcripts that can, in part, avoid reiterative transcription and be elongated normally. In this study, we examined the regulatory requirement for the long T · A tract in the upp initially transcribed region. We constructed upp promoter mutations that shorten the T · A tract to 7, 6, 5, 4, 3, or 2 bp and examined the effects of these mutations on upp expression and regulation. The results indicate that pyrimidine-mediated regulation is gradually reduced as the T · A tract is shortened from 7 to 3 bp; at which point regulation ceases. This reduction in regulation is due to large-percentage increases in upp expression in cells grown under conditions of pyrimidine excess. Quantitation of cellular transcripts and in vitro transcription studies indicate that the observed effects of a shortened T · A tract on upp expression and regulation are due to increases in the fraction of both G6- and A7-initiated transcripts that avoid reiterative transcription and are elongated normally.