A previous report [Motojima, K., Yamato, I. & Anraku, Y. (1978) J. Bacteriol. 136, 5-9) described the characteristics of mutants (proT-) of Escherichia coli K-12 that are defective in proline transport carrier activity. Two hybrid plasmids from the Clarke and Carbon colony bank were found to complement the mutation by conjugation and transformation. Analysis with restriction endonucleases showed that both plasmid DNAs carried the same fragment of the E. coli chromosome. A polypeptide with a molecular weight of 24,000, specifically coded for by the proT+ plasmid, was identified in the cytoplasmic membrane by using a double-isotope labeling method in a minicell system. The strain harboring the proT+ plasmid has 6 times as much proline transport carrier as the wild strain. This amplification of the carrier makes it possible to measure proline binding to the carrier by microequilibrium dialysis. Detailed analysis of binding indicated that the maximal amount of proline bound to the carrier is 0.70 nmol/mg of protein of the cytoplasmic membrane of the amplified strain. From this value and the assumption that the carrier has one binding site per minimal molecular weight of 24,000, the content of the proline transport carrier in the cytoplasmic membrane was estimated to be 1.7%.