The direction of transcription of the carAB gene cluster, which codes for Escherichia coli carbamoylphosphate synthase, was deduced from the effects of phage Mu-1 insertions in each of the two genes and from the results of ribonucleic acid-deoxyribonucleic acid hybridization experiments relating the quantity of car messenger ribonucleic acid to the location of various car mutations. The car locus appears to constitute an operon polarized from carA to carB. The levels of carA and carB products were determined in a large number of car mutants by using in vitro and in vivo complementation assays. The results obtained display strong anomalies, which are discussed in light of the conclusions described above.