Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr73, Tyr89, Tyr90, Glu204, Tyr235, Asp303, and Tyr423 were strongly involved in both uptake and excretion and that Tyr55, Glu76, Tyr246, Tyr310, Cys370, and Glu377 were moderately involved in both activities. Mutations of Trp43, Tyr57, Tyr107, Tyr366, and Tyr368 mainly affected uptake activity, and Trp41, Tyr174, Asp185, and Glu408 had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the Km value. Mutation of Arg299 mainly affected excretion, suggesting that Arg299 is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys370 seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys125, Cys389, or Cys394 together with Cys370. The relative topology of twelve transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys370. The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI and XII.