This study was conducted mainly to investigate the relative contributions of various mechanisms by which bile salts and EDTA may improve the in vitro rectal penetration of insulin in the albino rabbit. Insulin could not cross the rectal mucosa unless Na glycocholate or other penetration enhancers were present. Penetration enhancement was attributed primarily to Na glycocholate's ability to reduce the barrier function of the rectal membrane and to increase the fraction of insulin in its monomeric form, and secondarily to Na glycocholate's ability to protect insulin from proteolysis. Na glycocholate was more effective than Na taurocholate, but less effective than Na deoxycholate and polyoxyethylene-9-lauryl ether in enhancing rectal insulin penetration. Although EDTA at 0.01 and 0.1% did not affect rectal insulin penetration, it augmented the penetration enhancement effect of 1% Na glycocholate without causing additional damage to the rectal membrane, as judged by protein release. Such an action was attributed to the synergistic effect associated with: 1) an increase in the permeability of the paracellular pathway by EDTA and 2) an increase in the proportion of insulin in the monomeric form by Na glycocholate. Results from parallel in vivo experiments have indicated that it may be possible to achieve significant penetration enhancement by using a combination of otherwise membrane-damaging penetration enhancers which act by complementary mechanisms at concentrations that are both effective and well tolerated by mucosal epithelial cells.