In human, vitamin C (L-ascorbic acid) is an essential micronutrient required for an array of biological functions including enzymatic reactions and antioxidation. We describe here the molecular cloning of a novel human cDNA encoding a vitamin C transporter SVCT1. SVCT1 is largely confined to bulk-transporting epithelia (e.g., kidney and small intestine) with a putative alternative-splice product present in thymus, Applying radiotracer and voltage-clamp approaches in cRNA-injected Xenopus oocytes, we found that SVCT1 mediates saturable, concentrative, high-affinity L-ascorbic acid transport (K-0.5 = 50-100 mu M) that is electrogenic and can be inhibited by phloretin, SVCT1 displays exquisite substrate selectivity, greatly favoring L-ascorbic acid over its isomers D-isoascorbic acid and dehydroascorbic acid and 2- or 6-substituted analogues, whereas glucose and nucleobases are excluded. We have mapped the SLC23A2 gene (coding for SVCT1) to human chromosome 5 in band 5q31.2-31.3, within a region commonly deleted in malignant myeloid (leukemia) diseases. In addition, we have demonstrated that the human SLC23A1 gene product is a related high-affinity L-ascorbic acid transporter (SVCT2) that is widely distributed in brain, retina, and a host of endocrine and neuroendocrine tissues. The molecular identification of the human L-ascorbic acid transporters now provides the tools with which to investigate their roles in vitamin C metabolism in health and disease.