The pyrBI operon of Escherichia coli K-12 encodes the subunits of the pyrimidine biosynthetic enzyme aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 220.127.116.11). Expression of this operon apparently is negatively regulated by the intracellular levels of UTP. To elucidate the regulatory mechanism in which UTP functions, the nucleotide sequence of the promoter-regulatory region of the pyrBI operon was determined and DNA fragments containing this region were transcribed in vitro. These experiments revealed a rho-independent transcriptional terminator (attenuator) located only 23 base pairs before the promoter-proximal end of the structural genes. Transcription initiated upstream at either of two potential pyrBI promoters was efficiently (approximately equal to 98%) terminated at this site, indicating that the regulation of pyrBI expression involves attenuation control. Additional features identified suggest a model for regulation in which the relative rates of UTP-dependent transcription within the pyrBI leader region and coupled translation of the leader transcript control transcriptional termination at the attenuator.