The objective of this study was to determine (a) the rate of hydrolysis of insulin and proinsulin in homogenates of various non-oral absorptive mucosae relative to the ileal mucosa, and (b) the inhibitory effect of various protease inhibitors on the degradation of these peptides. Overall, insulin was somewhat more susceptible to hydrolysis than proinsulin in all mucosal homogenates. Proteolytic activity was highest in the nasal and rectal homogenates, followed by the ileal, vaginal, conjunctival and buccal homogenates in that order. The rate of insulin and proinsulin proteolysis differed by a factor of about 6 to 7 between the least and the most proteolytically active mucosa. A 5-fold maximum reduction in proteolytic rate was seen in the presence of Na glycocholate, aprotinin and p-chloromercuriphenyl-sulfonic acid (PCMPS). The rank order of effectiveness was Na glycocholate (1%) greater than aprotinin greater than PCMPS in all but the conjunctival and vaginal homogenates. Extrapolating the above in vitro findings to in vivo, both insulin and proinsulin are expected to be degraded to varying degrees during absorption across the various mucosae. Absorption may therefore be enhanced in the presence of protease inhibitors such as aprotinin, PCMPS, or Na glycocholate.