The purpose of this study was to evaluate the effectiveness and the toxicity of polyunsaturated fatty acid, such as oleic acid, eicosapentaenoic acid (DHA), as potential absorption enhancer for rectal delivery of insulin, using a water-in-oil-in water (W/O/W) multiple emulsion. In a single administration study, rectal insulin absorption was enhanced markedly, and marked hypoglycemia was induced by the emulsion incorporating various fatty acids in an insulin dose-related fashion. The pharmacological availability of the emulsion incorporating 2% oleic acid, EPA and DHA was approximately 7.7, 11.0 and 25.4%, respectively. The insulin absorption enhancement effect was not increased in proportion to the amount of DHA in the emulsion, the mean T(max) value of the serum glucose-time curve could be extended to twice that of the emulsion without PF 127. In a multiple administration study, the mean AUC(glucose) values of the emulsion incorporating DHA showed almost the same value on the first and the tenth day. From the morphological appearance of the mucosal surface, the emulsion incorporating DHA induced no or little mucosal damage. Our findings demonstrated that DHA has a strong insulin permeability enhancement effect and little toxicity. Thus, DHA is an attractive candidate as an absorption enhancer for intestinal delivery of insulin.