Protein synthesis in 'minicells' showed that the DNA immediately preceding the pyrE gene of Escherichia coli directs the formation of considerable amounts of a polypeptide (mol. wt. approximately 30 000) of unknown function. The nucleotide sequence of this DNA revealed the existence of an open reading frame (ORF) of 238 codons that ends 68 nucleotide residues upstream to the structure start of pyrE, just prior to the GC-rich symmetry region of a sequence with features characteristic of a rho-independent transcription terminator. Deletion of the start of this 238 codons long ORF gene resulted in a dramatic fall in the level of pyrE expression, indicating that the two genes (ORF and pyrE) constitute an operon. S1-nuclease digestion of RNA-DNA hybrids revealed that both genes are transcribed from two promoters (P1 and P2) located in front of the ORF start. Furthermore, when the RNA used in these experiments was prepared from cells with different levels of pyrE expression, created by manipulations in their pyrimidine nucleotide supply, the frequency of transcription initiations at P1 and P2 was found to be constitutive, while only a pyrimidine regulated fraction of the mRNA chains reached into the pyrE gene. In vitro transcription of isolated DNA fragments showed that the mRNA chains are terminated between the ORF gene and pyrE. From these observations we conclude that pyrE expression is controlled by a UTP modulated intercistronic attentuation.