The kdsB gene from Escherichia coli K-12, which encodes CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase), was cloned into pBR322 as an 8-kilobase PstI fragment. Selection of this cloned segment was facilitated by using Salmonella typhimurium SL5283, which is deficient in three restriction enzyme systems and thus allows efficient cloning of E. coli DNA in S. typhimurium. The temperature-sensitive kdsB gene from S. typhimurium HD2 was transduced into strain SL5283 after the insertion of transposon Tn10 near the kdsB allele. Tetracycline-sensitive variants of strain SL5283 were then derived and used to select clones of the E. coli K-12 gene, inserted into the PstI site of pBR322, by complementation of the temperature-sensitive lesion in kdsB. One plasmid, pRG-1, complemented the kdsB temperature-sensitive allele and had the following characteristics: (i) it coded for several polypeptides by coupled transcription-translation in vitro, including one polypeptide which comigrated with CMP-KDO synthetase during polyacrylamide gel electrophoresis in sodium dodecyl sulfate; (ii) it overproduced CMP-KDO synthetase activity 20- to 40-fold depending on strain and growth conditions; and (iii) it coded for activity of CMP-KDO synthetase which, when purified to homogeneity, had the same molecular weight and kinetic characteristics as CMP-KDO synthetase of chromosomal origin.