Protease inhibition has been postulated to be one of the several mechanisms by which penetration enhancers promote the mucosal absorption of peptide and protein drugs. The objective of this study was to determine whether protease inhibition by Na glycocholate and polyoxyethylene-9-lauryl ether, two extensively studied enhancers, led to suppression of insulin proteolysis over a range of insulin concentrations. To this end, the rate of insulin proteolysis in nasal tissue supernatants of the albino rabbit was determined in the presence of 0.1-2% Na glycocholate and polyoxyethylene-9-lauryl ether and at insulin concentrations ranging from 5 to 100 microM. Partly due to self-association, insulin was self-stabilizing against nasal proteolysis as its concentration was raised from 5 to 100 microM. At insulin concentrations lower than 50 microM, both Na glycocholate and polyoxyethylene-9-lauryl ether reduced the rate of insulin proteolysis. By contrast, at 100 microM insulin concentration, both enhancers accelerated insulin proteolysis. Such an effect was attributed to the deaggregation of insulin by the enhancers, increasing the proportion of monomers available for nasal proteolysis. The incorporation of 0.1 mM PCMPS, a potent inhibitor of insulin proteolysis, partly overcame the accelerating effect of Na glycocholate on insulin proteolysis.