The nucleotide sequence of the operon for the putrescine transport system that maps at 19 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding potF, -G, -H, and -I proteins. The potF protein (M(r) = 38,000) was inferred to be a putrescine-specific binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. The potG protein (M(r) = 45,000) had consensus amino acid sequences for the nucleotide-binding site. The potH (M(r) = 35,000) and potI (M(r) = 31,000) proteins consisted of six putative transmembrane-spanning segments linked by hydrophilic segments of variable length as shown by hydropathy profiles. The spermidine-putrescine transport system, which is mainly involved in spermidine transport, consisted of potA, -B, -C, and -D proteins (Furuchi, T., Kashiwagi, K., Kobayashi, H., and Igarashi, K. (1991) J. Biol. Chem. 266, 20928-20933). The homologies of the corresponding two proteins between those two systems, F and D, G and A, H and B, and I and C, were 35, 42, 37, and 36%, respectively. The initiation point of the transcription of the operon for the putrescine transport system was determined by primer extension and S1 nuclease mapping. Transcription started from the T residue located either 149 or 150 nucleotides upstream from the initiator AUG codon of potF protein mRNA. By making several subclones and a mutant lacking the potF gene, we showed that the expression of all four proteins was necessary for maximal putrescine transport activity. These results indicate that the putrescine transport system can also be defined as a bacterial periplasmic transport system.