We have cloned a Na ؉ -dependent, high affinity dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na ؉ -dependent, low affinity dicarboxylate transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na ؉ -dependent transport of succinate with a K t of 2 M. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na ؉ concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li ؉ inhibits NaDC3 in the presence of Na ؉ . Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8.0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6.5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na ؉