The pyrC gene of Salmonella typhimurium, encoding the third enzyme of pyrimidine nucleotide biosynthesis, dihydroorotase, has been cloned into the multicopy plasmid pBR322. The recombinant plasmid, pJRC1, promoted the synthesis of 20-30-fold elevated levels of dihydroorotase. The expression of pyrC was regulated to the same extent by pyrimidines whether present on the multicopy plasmid or in single copy on the chromosome. A comparison of the polypeptides encoded by pyrC-complementing and non-complementing plasmids showed the gene product to have a molecular mass of approximately 37 kDa. The nucleotide sequence of the gene and 400 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 38 500 Da, was deduced to be the coding region for pyrC. S1 nuclease mapping indicated that transcription of pyrC is initiated 40 base pairs upstream from the translational start. Subcloning of a 184-base-pair DNA fragment, which included 118 base pairs upstream from the transcriptional start, and the first eight codons of the pyrC structural gene, into a galK expression vector, established that the pyrC promoter and regulatory region are harbored on this fragment. The leader region does not show any features resembling the attenuators found in front of the coding regions of pyrB and pyrE; however, it contains a region of dyad symmetry, which may allow the leader transcript to form a stable hairpin. The possible significance of this putative hairpin formation in the regulation of pyrC expression is discussed.