The carAB operon of Salmonella typhimurium encoding carbamoyl-phosphate synthetase (CPSase) has been cloned, and the nucleotide sequence of the first gene of the operon, carA, together with 760 base pairs of the 5'-flanking region was determined. The product of the carA gene is the small subunit of CPSase. It catalyzes the transfer of the amide group from glutamine to an NH3-site on the heavy subunit. Primer extension and S1 nuclease mapping of in vivo carAB transcripts revealed that transcription is similar to that of Escherichia coli [Piette, J. et al. (1984) Proc. Natl Acad. Sci. USA 81, 4134-4138] in its initiation at two promoters, P1 and P2, controlled by pyrimidines and arginine, respectively. The arginine control is mediated through binding to the arginine repressor (argR). The involvement of titratable regulatory elements is indicated by the escape from both arginine and pyrimidine control, when the operon is present in multicopies on a plasmid. Measurements of CPSase levels in mutants which allows independent manipulation of the intracellular uracil and cytosine nucleotide pools show, that both uracil and cytosine nucleotides are required for full repression and that limitation of either nucleotide results in derepression of CPSase synthesis. Deletion analyses indicate that regions upstream of the P1 promoter are required for normal expression from this promoter but not from P2.