The absorption of glycerol was examined using the closed loop of the rat small intestine in situ to clarify the transport mechanism. The absorption of glycerol, evaluated by its disappearance from the intestinal lumen, was saturable and reduced under the Na⁺-free conditions, suggesting the involvement of an Na⁺-dependent carrier-mediated transport system. Furthermore, glycerol absorption was selectively inhibited by several alcohols, among which 1,3-propanediol caused the greatest inhibition, and also by glycerol-3-phosphate and voglibose, which are alcohol-related compounds analogous to glycerol. Several other compounds that did not inhibit glycerol absorption included D-glucose and L-ascorbate, which are known to be transported by specific carriers. Therefore, the carriers for these two compounds do not seem to be involved in glycerol absorption. It is likely that the carrier-mediated transport system involved in glycerol absorption is specific to glycerol and, possibly, some analogous compounds with hydroxyl groups. Thus, the present study has provided in situ evidence for the presence of an Na⁺-dependent carrier-mediated transport system for glycerol in the rat small intestine. It would be interesting to examine the possibility that the carrier-mediated glycerol transport system could be involved in drug absorption and also that it could be used for oral drug delivery.