A deoxyribonucleic acid-directed in vitro system for the synthesis of Escherichia coli carbamoylphosphate synthase has been developed, and its properties have been studied. The system uses the deoxyribonucleic acid of a lambda phage carrying the car genes (lambdadcarAB) as template and mediates the synthesis of both subunits of the enzyme. This newly synthesized enzyme exhibits the properties of native carbamoylphosphate synthase. A study of the in vitro synthetic capacities of S-30 extracts from strains containing either a mutated or the wild-type allele of gene argR supports earlier suggestions, based on in vivo evidence, that the argR product is involved in cumulative repression of carbamoylphosphate synthase by arginine and the pyrimidines. Repression in vitro is as efficient as in vivo. In keeping with such observation it is shown that in vitro synthesis of carbamoylphosphate synthase is repressed by partially purified arginine repressor. Evidence was obtained which indicates that arginine repression of carbamoylphosphate synthase mainly operates at the level of transcription. This was based on the design of an in vitro transcription system for gene carA, the structural gene for the light subunit of carbamoylphosphate synthase. This system also allowed us to demonstrate that free arginine is the corepressor involved in carbamoylphosphate synthase repression. The present in vitro approaches, in addition to the information they have already provided, open new possibilities for further investigations on the mechanism of cumulative repression and, in particular, on the participation of pyrimidine end products in this regulatory mechanism.