Functions of potA and -D proteins in the spermidine-preferential uptake system, which consists of potA, -B, -C, and -D proteins, were studied. Spermidine uptake activity was lost when the gene for potA or potD protein was disrupted, and transformation of the cells with potA or potD gene recovered the uptake activity. PotD protein was found to bind spermidine with a 3.2 microM dissociation constant. Spermidine uptake by membrane vesicles prepared from Escherichia coli DR112 containing the genes for potA, -B, and -C proteins was strongly dependent on the addition of potD protein, and its optimal concentration was 5 microM when 10 microM spermidine was used as substrate. The ATP dependence of spermidine uptake was examined with the atp mutant of E. coli. The uptake was completely dependent on ATP. When the membrane potential was extinguished by carbonyl cyanide m-chlorophenylhydrazone, the uptake activity was decreased by 60% even if ATP existed. This suggests that the membrane potential is also involved in the spermidine uptake. ATP was found to bind to potA protein. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of potA protein, which is located between two consensus amino acid sequences for nucleotide binding, was replaced by methionine. Although the amount of mutated potA protein expressed in E. coli cells was the same as that of normal potA protein and the mutated protein was membrane-associated, no significant spermidine uptake was observed. The results taken together indicate that potA and -D proteins are absolutely necessary for spermidine uptake in conjunction with the two channel forming proteins (potB and -C).