The ATPase activity of PotA, a component of the spermidine-preferential uptake system consisting of PotA, -B, -C, and -D, was studied using purified PotA and a PotABC complex on inside-out membrane vesicles. It was found that PotA can form a dimer by disulfide cross-linking but that each PotA molecule functions independently. When PotA was associated with the membrane proteins PotB and PotC, the K(m) value for ATP increased and PotA became much more sensitive to inhibition by spermidine. It was also shown that spermidine uptake in cells was gradually inhibited in parallel with spermidine accumulation in cells. The results suggest that spermidine functions as a feedback inhibitor of spermidine transport. The function of PotA was analyzed using PotA mutants obtained by random mutagenesis. There are two domains in PotA. The NH2-terminal domain (residues 1-250) contains the ATP binding pocket formed in part by residues Cys26, Phe27, Phe45, Cys54, Leu60, and Leu76, the active center of ATPase that includes Val135 and Asp172, and amino acid residues necessary for the interaction with a second PotA subunit (Cys26) and with PotB (Cys54). The COOH-terminal domain (residues 251-378) of PotA contains a site that regulates ATPase activity and a site involved in the spermidine inhibition of ATPase activity.