The transport of riboflavin across the brush border membrane of human intestine was examined using the established brush border membrane vesicle technique. Both osmolarity and temperature studies have concluded that the uptake of riboflavin by these vesicles is mostly the result of transport of riboflavin into an active intravesicular space with less binding to membrane surfaces. When an inwardly directed Na+ gradient was imposed, transport of riboflavin was linear with time for approximately 20 seconds of incubation and was significantly higher than in the presence of an identical K+ gradient. Initial rate of transport of riboflavin as a function of concentration was found to include a saturable component in the presence of an inwardly directed Na+ gradient but was linear in the presence of an identical K+ gradient. The apparent Km and Vmax of the Na+ stimulated transport process were found to be 7.26 mumols/L and 0.97 pmol/mg protein per 10 seconds, respectively. The addition of high concentrations of unlabeled riboflavin and its structural analogue lumiflavin to the incubation medium caused significant inhibition in the transport of 3H-riboflavin in the brush border membrane vesicle incubated in the presence of an inwardly directed Na+ gradient but not in vesicles incubated in the presence of an identical K+ gradient. Inducing a relatively positive intravesicular space with the use of valinomycin and an inwardly directed K+ gradient caused significant inhibition in the transport of riboflavin. On the other hand, inducing a relatively negative intravesicular space with the use of anions of different lipid permeabilities caused significant stimulation in the transport of riboflavin. These results demonstrate that riboflavin transport in human intestinal brush border membrane vesicle is through a carrier-mediated system. This system functions in the presence of a Na(+)-gradient and seems to transport the substrate by an electrogenic process.