The present study was aimed at kinetically characterizing the newly found carrier-mediated riboflavin transport system in the rat colon, comparing it with that in the small intestine, and also probing the potential roles of these transport systems in intestinal drug absorption. Riboflavin transport, evaluated by measuring the initial uptake into everted intestinal tissue sacs, was saturable with a Michaelis constant (Km) of 0.13 microM and a maximum transport rate (Jmax) of 0.74 pmol/min/100 mg wet tissue weight (wtw) in the colon. Both the Km and the Jmax were smaller than those (0.57 microM and 4.26 pmol/min/100 mg wtw, respectively) in the small intestine, suggesting that the transport system in the colon has a higher affinity to substrates and a smaller transport capacity than its counterpart in the small intestine. The carrier-mediated riboflavin transport in the colon, similarly to that in the small intestine, was Na+-dependent and inhibited by lumiflavin, a riboflavin analogue with an isoalloxazine ring, but not by D-ribose, which forms the side-chain attached to the isoalloxazine ring in riboflavin. To further clarify the substrate specificities of the transport systems, we examined the effects of several drugs with a tricyclic structure similar to isoalloxazine ring on riboflavin transport. Chlorpromazine, a phenothiazine derivative, was found to inhibit riboflavin transport in both the small intestine and the colon. Methylene blue also was found to be a potent inhibitor in both sites. These results suggest that some tricyclic-type drugs could interfere with intestinal riboflavin absorption by specific carrier-mediated transport systems. These transport systems may play roles in the absorption of tricyclic-type drugs.