Previous Recommended Dietary Allowances (RDAs) for vitamin C (ascorbate) and other water-soluble vitamins were based on preventing deficiency diseases with a margin of safety. We proposed that new RDAs for vitamins, with vitamin C as a model vitamin, could be determined using in situ kinetics, a concept developed by this laboratory. This proposal was adopted and expanded by the National Academy of Sciences as part of revised recommendations for vitamin C intake released in 2000. In situ kinetics has biochemical and clinical goals. Biochemical goals are to determine vitamin molecular functions in relation to vitamin concentrations. For vitamin C studies its function is investigated in human tissues such as fibroblasts and neutrophils. To determine how intracellular concentration is regulated, two pathways of vitamin C accumulation were characterized. In the first pathway, vitamin C is transported as such by two carriers that are sodium-dependent, saturable, energy dependent, and inhibited by laboratory-synthesized ascorbate analogs. The two human transporters hSVCT1 and hSVCT2 were cloned and characterized, and genomic characterization and studies of nucleotide polymorphisms are underway. Created mice deficient in the transporter SVCT2 did not survive the perinatal period and had very low or undetectable vitamin C concentrations in many but not all tissues, indicating that vitamin C as such is the dominant transported species. In the second pathway the extracellular oxidized form of vitamin C, dehydroascorbic acid, is accumulated as ascorbate within neutrophils by the process of ascorbate recycling. Oxidants from neutrophils oxidize extracellular vitamin C to dehydroascorbic acid. Dehydroascorbic acid is transported by facilitative glucose transporters GLUT I, III, and IV, and immediately reduced intracellularly to ascorbate by glutaredoxin (thioltransferase). Glutaredoxin from neutrophils was isolated, identified as the reducing activity, cloned, and characterized. Our studies show that vitamin C recycling occurs in neutrophils when activated by bacteria, and only in neutrophils and not in bacteria. Studies are ongoing to characterize potential roles of vitamin C in neutrophils, including oxidant quenching, bacterial killing, and regulation of neutrophil apoptosis. Overall findings suggest that vitamin C function can be determined in relation to its concentration in living tissues. Clinical goals of in situ kinetics are to determine how vitamin concentrations are achieved in normal humans as a function of dose and whether achieved concentrations have functional consequences. Extensive clinical studies were undertaken in healthy men and women inpatients hospitalized at the Clinical Center. For the first time, the following were described: the relationship between vitamin C doses over a wide range and its concentrations in plasma and tissues; true bioavailability of vitamin C; vitamin urinary excretion in relation to dose; functional antioxidant consequences of vitamin C in vivo, a three component pharmacokinetics model of vitamin C distribution in humans, and potential adverse effects in relation to dose. Continuing analyses of the extensive data generated from these studies are ongoing. Based on our data to date, RDAs for vitamin C in the U.S. and Canada were revised upward in 2000 by the National Academy of Sciences and were also increased in the following countries: Germany, Austria, Denmark, France, Japan, and China. Because the known health benefits from vitamin C are from foods containing the vitamin, we recommend that vitamin C intake is from at least 5 servings of fruits and vegetables daily. Forthcoming molecular and clinical data from our laboratory will have further impact on vitamin C intake recommendations for healthy and ill people.