The stability of insulin and its acyl derivatives in the small intestine was examined in vitro. When these compounds were incubated in small intestinal fluid at 37 degrees C, proteolysis of monoacyl insulins was reduced by increasing the carbon number of the fatty acid attached to Phe-B1 of the insulin molecule. In contrast, Phe-B1 and Lys-B29 diacylated insulins were more susceptible to hydrolysis than native insulin. Similar results were obtained using homogenates of the small intestinal mucosa, although the extent of the contribution of acylation to insulin degradation differed. The mechanism of the accelerated insulin proteolysis by diacylation was studied by circular dichroism (CD). The negative maxima at 270 nm in the CD spectra were attenuated for the diacyl derivatives, indicating that insulin association was inhibited by diacylation. Therefore, the increased proportion of monomers available for insulin proteolysis represents a main factor that makes diacyl derivatives unstable.