The predicted secondary structure model of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) presents the 12-transmembrane helix motif observed for many secondary transporters. Biochemical evidence presented in this paper is not consistent with this model. N-terminal and C-terminal fusions of CitS with the biotin acceptor domain of the oxaloacetate decarboxylase of K. pneumoniae catalyze citrate transport, showing the correct folding of the CitS part of the fusion proteins in the membrane. Proteolysis experiments with these fusion proteins revealed that the N terminus of CitS is located in the cytoplasm, while the C terminus faces the periplasm. The membrane topology was studied further by constructing a set of 20 different fusions of N-terminal fragments of the citrate transporter with the reporter enzyme alkaline phosphatase (CitS-PhoA fusions). Most fusion points were selected in hydrophilic areas flanking the putative transmembrane-spanning domains in CitS that are predicted from the hydropathy profile of the primary sequence. The alkaline phosphatase activities of cells expressing the CitS-PhoA fusions suggest that the polypeptide traverses the membrane nine times and that the C-terminal half of the protein is characterized by two large hydrophobic periplasmic loops and two large hydrophilic cytoplasmic loops. CitS belongs to the family of the 2-hydroxycarboxylate transporters in which also the citrate carriers, CitPs, of lactic acid bacteria and the malate transporter, MleP, of Lactococcus lactis are found. Since the hydrophobicity profile of CitS is very similar to the hydrophobicity profiles of CitP and MleP, it is most likely that the new structural motif of nine transmembrane segments is shared within this new transporter family.