Our previous experience with a continuous flow-through perfusion system has demonstrated its usefulness for studying diffusion kinetics of drugs across small intestinal mucosa for bioavailability/bioequivalence (BA/BE) studies. During the last decade, delivery of drugs to the colon for systemic absorption as well as for local delivery in certain colonic diseases, has been extensively investigated. For this reason, we sought to assess the in vitro comparative permeability of human vaginal, small intestinal and colonic mucosa using a flow-through perfusion method. It was clear from our studies that human colonic epithelium was statistically significantly (P<0.05) more permeable to water, 17β-estradiol, arecoline and arecaidine than intestinal mucosa. However, both these mucosae were statistically significantly less permeable to the above four permeants than human vaginal mucosa. As previously shown for small intestinal mucosa, the low in vitro permeability of colonic mucosa to drugs with molecular weight >300Da may necessitate using other epithelial membranes, e.g. vaginal mucosa, as alternative barriers for in vitro BA/BE studies. We also concluded that the flow-through mucosal perfusion system used in our laboratory is therefore also potentially useful for determining the permeability of a therapeutic agent from the colon for registration purposes.