Isolated intestinal mucosa cells of the guinea pig were employed to study the intestinal transport of riboflavin. This method provided a direct approach to characterize the mechanism of riboflavin absorption. Riboflavin was taken up by both jejunal and ileal cells in a temperature-dependent process. Uptake was energy-dependent and exhibited saturation kinetics with an apparent Km ranging between 70 and 87 nM and Vmax of 1.6 and 1.82 pmol/(mg cell protein x minute) for jejunal and ileal cells, respectively. Absence of Na+ or presence of ouabain in the incubation medium strongly decreased the rate and extent of transport. Riboflavin uptake was inhibited by the flavins FAD and FMN, but not by ribose. These results indicate that riboflavin is absorbed in all parts of the small intestine in an active, carrier-mediated process.