In the present study, we determined the functional role of fifteen positively charged amino acid residues at or within one of the predicted eleven transmembrane helices of the flounder renal sodium/dicarboxylate cotransporter, fNaDC-3. Using site-directed mutagenesis, histidine (H), lysine (K) and arginine (R) residues of fNaDC-3 were replaced by alanine (A), isoleucine (I), or leucine (L). Most mutants showed sodium-dependent, lithium-inhibitable [14C]succinate uptake and, in two-electrode voltage-clamp (TEVC) experiments, Km and Imax values comparable to wildtype (wt) fNaDC-3. The replacement of R109 and R110 by alanine and isoleucine (RR109/110AI) prevented the expression of fNaDC-3 at the plasma membrane. When the lysines at positions 232 and 235 were replaced by isoleucine (KK232/235II), the transporter was expressed, but showed small transport rates and succinate-induced currents. K114I, located within transmembrane helix 4, showed [14C]succinate uptake similar to the wt, but relatively small inward currents. When K114 was replaced by arginine, glutamic acid (E), or glutamine (Q), all mutants were expressed at the cell surface. In [14C]succinate uptake and TEVC experiments performed simultaneously on the same oocytes, uptake was similar to or higher than wt, whereas succinate-induced currents were either comparable (K114R) to, or considerably smaller (K114E, K114I, K114Q) than, those evoked by the wt. These results suggest that a positively charged residue at position 114 is required for electrogenic sodium/dicarboxylate cotransport.