We have examined the in-vitro permeability characteristics of insulin in the presence of various absorption enhancers across rat intestinal membranes and have assessed the intestinal toxicity of the enhancers using an in-vitro Ussing chamber method. The absorption enhancing mechanism of n-lauryl-beta-D-maltopyranoside was studied also. The permeability of insulin across the intestinal membranes was low in the absence of absorption enhancers. However, the permeability was improved in the presence of enhancers such as sodium glycocholate and sodium deoxycholate in the jejunum, and sodium glycocholate, sodium deoxycholate, n-lauryl-beta-D-maltopyranoside, sodium caprate and ethylenediaminetetraacetic acid (EDTA) in the colon. Overall, the absorption enhancing effects were greater on the colonic membrane than on the jejunal membrane. The intestinal membrane toxicity of these enhancers was characterized using the release of cytosolic lactate dehydrogenase from the colonic membrane. A marked increase in the release of lactate dehydrogenase was observed in the presence of sodium deoxycholate and EDTA. The release of lactate dehydrogenase in the presence of these absorption enhancers was similar to that seen with sodium dodecyl sulphate (SDS), used as a positive control, indicating high toxicity of these enhancers to the intestinal membrane. In contrast, sodium glycocholate and sodium caprate caused minor releases of lactate dehydrogenase, similar to control levels, suggesting low toxicity. In addition, the amount of lactate dehydrogenase in the presence of n-lauryl-beta-D-maltopyranoside was much less than that seen with sodium deoxycholate, EDTA and SDS. Therefore, sodium glycocholate, sodium caprate and n-lauryl-beta-D-maltopyranoside are useful absorption enhancers due to their high absorption enhancing effects and low intestinal toxicity. To investigate the absorption enhancing mechanisms of n-lauryl-beta-D-maltopyranoside, the transepithelial electrical resistance (TEER), voltage clamp experiments and the circular dichroism spectra were studied. n-Lauryl-beta-D-maltopyranoside decreased the TEER values in a dose-dependent manner, suggesting that the enhancer may open the tight junctions of the epithelium, thereby increasing the permeability of insulin via a paracellular pathway. This speculation was supported by the findings that 20 mM n-lauryl-beta-D-maltopyranoside produced a greater increase in the paracellular flux rate than in the transcellular flux rate by the voltage clamp studies. Evaluating the circular dichroism spectra we found that insulin oligomers were not dissociated to monomers by the addition of n-lauryl-beta-D-maltopyranoside, but dissociation did occur with the addition of sodium glycocholate. Thus, the dissociation of insulin was not a major factor in the absorption enhancing effect of n-lauryl-beta-D-maltopyranoside. These findings provide basic information to select the optimal enhancer for the intestinal delivery of peptide and protein drugs including insulin.