Cell-penetrating peptides such as HIV-1 Tat and oligoarginine are attractive tools for the intracellular delivery of therapeutic macromolecules. Although we have found that oligoarginine enhances the intestinal absorption of therapeutic peptides, especially insulin, the mechanism underlying the ability of oligoarginine to increase intestinal drug absorption is unclear. In addition, there is no information about the permeation characteristics of these functional peptides through the biological intestinal membrane. Therefore, in this study the permeation characteristics of oligoarginine itself across the intestinal membrane were first determined to obtain the information about absorption enhancement mechanisms. Incubation at low temperature and coincubation with heparin reduced the tissue distribution and permeation of fluorescein-labeled oligoarginine (FL-d-R6) through the rat ileal membrane. These results suggest that the attachment of FL-d-R6 to cell-surface proteoglycans and energy-dependent endocytosis are involved in its permeation through the ileal epithelial membrane. Based on the characteristics of oligoarginine, we attempted to facilitate the intestinal permeation of the peptide drug, leuprolide, using the function of oligoarginine. However, leuprolide permeation was not achieved when leuprolide was applied with oligoarginine to mucosal side of rat ileal sheets or when a leuprolide-oligoarginine conjugate was administered. These findings emphasize that any strategy using oligoarginine to improve intestinal drug permeation requires an intermolecular interaction, such as an electrostatic interaction, and a covalent linkage between the macromolecular drug and oligoarginine may hamper the ability of oligoarginine to enhance intestinal epithelial permeation of therapeutic peptides and proteins.