Normal microflora of the large intestine synthesize a number of water-soluble vitamins including riboflavin (RF). Recent studies have shown that colonic epithelial cells possess an efficient carrier-mediated mechanism for absorbing some of these micronutrients. The aim of the present study was to determine whether colonic cells also possess a carrier-mediated mechanism for RF uptake and, if so, to characterize this mechanism and study its cellular regulation. Confluent monolayers of the human-derived nontransformed colonic epithelial cells NCM460 and [(3)H]RF were used in the study. Uptake of RF was found to be 1) appreciable and temperature and energy dependent; 2) Na(+) independent; 3) saturable as a function of concentration with an apparent K(m) of 0.14 microM and V(max) of 3.29 pmol x mg protein(-1) x 3 min(-1); 4) inhibited by the structural analogs lumiflavin and lumichrome (K(i) of 1.8 and 14.1 microM, respectively) but not by the unrelated biotin; 5) inhibited in a competitive manner by the membrane transport inhibitor amiloride (K(i) = 0.86 mM) but not by furosemide, DIDS, or probenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RF uptake in RF-deficient and oversupplemented conditions, respectively; and 7) modulated by an intracellular Ca(2+)/calmodulin-mediated pathway. These studies demonstrate for the first time the existence of a specialized carrier-mediated mechanism for RF uptake in an in vitro cellular model system of human colonocytes. This mechanism appears to be regulated by extracellular substrate level and by an intracellular Ca(2+)/calmodulin-mediated pathway. It is suggested that the identified transport system may be involved in the absorption of bacterially synthesized RF in the large intestine and that this source of RF may contribute toward RF homeostasis, especially that of colonocytes.