Absorption characteristics of insulin derivatives chemically modified with various fatty acids in the intestine were determined by in situ loop and in vitro modified Ussing chamber methods. The pharmacological activities of these acyl derivatives, as assessed by their hypoglycemic effects after intravenous administration, were reduced upon increasing the carbon number of the fatty acid(s) chemically attached to native insulin. However, high pharmacological activities were seen when mono-and dicaproyl derivatives were administered intravenously. The absorption of insulin after its small intestinal administration could be hardly improved by acylation. In contrast, its absorption after the large intestinal administration was increased by increasing the number of caproic acid molecules attached to insulin. Furthermore, by an in vitro modified Ussing chamber method, it was revealed that the permeability of insulin across both the duodenal and colonic mucous membranes was also improved by increasing the number of caproic acid molecules. These in situ and in vitro results indicated that the chemical modification of insulin with fatty acids was a useful approach for improving insulin absorption from the large intestine.