Rectal administration of insulin as a suppository or p.o. administration of a proteolysis-resistant insulin analog lowers serum glucose levels, suggesting uptake and/or diffusion of intact insulin across the epithelium of the large intestine; however, no rigorous studies of insulin degradation and/or transepithelial flux in vitro have been reported with isolated colonic epithelium. Everted and noneverted sacs of rat distal colonic epithelium were prepared and incubated at 37°C with porcine [125I]insulin and less than 1% of the [125I]insulin crossed the epithelial barrier under these in vitro conditions. The apical surface degraded [125I]insulin at a rate of 0.071 fmol/mg of dry wt./min, whereas the submucosal surface degraded insulin at a rate of 0.045 fmol/mg of dry wt./min. Over 60% of the available [125I]insulin was degraded by the apical surface of the distal colon during a 15-min incubation, whereas approximately 40% of the radioligand was degraded by the submucosal surface under identical conditions. Degradation of [125I]insulin was inhibited partially by the addition of excess unlabeled insulin and inhibited completely that the mammalian colonic epithelium is an effective barrier to transepithelial flux of insulin and identify insulin-degrading activity on both the apical and submucosal surfaces of the colonic epithelium. Although the colonic epithelium represents a significant physiological barrier to the uptake of insulin from the intestinal lumen, paradoxically, administration of large doses of unlabeled insulin into the distal colon or into the entire large intestine caused a dose-dependent increase in serum insulin levels during a 15-min experiment in vivo.