The transport-enhancing effects of medium-chain fatty acids (caproic, caprylic, and capric acids) and their acylglycerols (mono-, di-, triacylglycerols) were investigated by using Caco-2 cell monolayers as a model of the human intestinal epithelium. Penicillin V was used as a model for a hydrophilic bioactive compound. Among the fatty acids and acylglycerols tested, 1,2-dicaproin, monocaprin, monocaprylin, and capric acid sodium salt effectively enhanced the transport rate, whereas other substances enhanced the rate only slightly or not at all. With each of these four substance, the rate of enhancement was proportional to the concentration at low concentrations, but leveled off at high concentrations. The transport-enhancing effects were well correlated with the reduction in surface tension and with a physico-chemical parameter, denoted by the surface energy-lowering coefficient, characterizing the surface activity of a substance.