The existence of a membrane-associated uptake carrier for riboflavin (RF) is demonstrated in Xenopus oocytes. Uptake of low (0.017 μM) and high (3 μM) concentrations of RF was linear with time for up to 2 hours, and occurred with little initial binding to oocytes, and little metabolism. Uptake of RF was found to be independent of extracellular pH and Na +. The initial rate of RF uptake was saturable as a function of concentration with an apparent K m of 0.41 ± 0.02 μM and a V max of 2.86 ± 0.04 fmol/oocyte per h. Uptake of 3H-RF was inhibited by unlabeled RF and by the structural analogs lumiflavin, isoriboflavin (iso-RF), 8-aminoriboflavin (8-NH 2-RF), 8-hydroxyriboflavin (8-OH-RF), and lumichrome, but was not affected by flavin adenine dinucleotide (FAD), d-ribose or lumazine. Uptake of RF was significantly retarded by the metabolic inhibitor 2,4-dinitrophenol. The sulfhydryl group-modifying reagents p-chloromercuriphenylsulfonate (pCMPS), p-chloromercuribenzoate (pCMB), N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) all caused significant inhibition in RF uptake. The inhibitory effect of pCMPS was completely reversed by treatment of pCMPS-pretreated cells with reducing agents. While the transmembrane transport inhibitors 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and furosemide had no effect on RF uptake, amiloride and probenecid suppressed RF uptake in a dose-dependent fashion. Closer examination of the inhibition mediated by amiloride showed that it was competitive in nature with an apparent K i of ∼ 1.8 mM, whereas the inhibition induced by probenecid was nonspecific. Together, these findings indicate that Xenopus oocytes possess an endogenous, specific, membrane-associated carrier-mediated uptake system for RF. The results also demonstrate the usefulness of Xenopus oocytes as a model system with which to study the RF transport event across biological membranes, which should further our present understanding of RF uptake by various vertebrate cells.