This study was designed to evaluate the percutaneous absorption of insulin in an attempt to develop an efficient transdermal therapeutic system (system) for the treatment of diabetes. First, the dissociation of porcine insulin existing mainly as hexamers was examined. Next, enhancement of the percutaneous absorption of the hormone was studied by the combined use of two or more kinds of enhancers which exert their enhancement effects by different mechanisms, or by preparing the liposomal formulation of insulin. Porcine insulin dissociated in 0.1 M glycine-HCl buffer (pH 4.0), probably to a dimer, this being demonstrated by the notable attenuation of the maxima at 221 and 274 nm of the circular dichroism (CD) spectra. Thus, 0.1 M (or partly 1 M) glycine-HCl buffer (pH 4.0) was selected for the preparation of all gel formulations. The in viv o absorption of insulin through Wistar rat skin was estimated by blood glucose level. System 3 containing liposomal insulin, D-limonen and taurocholate gave the greatest hypoglycemic response, out of the formulations used, with its response persisting over a 10 h period and resulting in the highest pharmacological availability (20.7±4.6%). The combination of n-octyl-β-D-thioglucoside (OTG), cineol and deoxycholate (system 6) or D-limonen and OTG (system 5) also produced a high hypoglycemic effect. The in vitro penetration of insulin was investigated using system 5 and 6. The percutaneous penetration of insulin was demonstrated by an in vitro experiment, but was small in quantity. Our data present unambiguous evidence that this hydrophilic macromolecule was absorbed through the stratum corneum of rat skin under selected conditions.