Abstract: AIM To evaluate the effect of proteolytic enzymes on the absorption of insulin in the buccal mucosa. METHODS The trichloroacetic acid method was used to estimate the degradation of insulin under different conditions in the buccal mucosal homogenates. In vivo experiments estimating the enhancement of hypoglycaemic effect by enzyme inhibitors were also conducted. RESULTS Proteolytic enzymes in the buccal mucosa were less active than in the intestine. Bacitracin, aprotinin and sodium deoxycholate can inhibit the degradation of insulin in the buccal mucosal homogenates. The degradation of insulin in buccal mucosal homogenates of normal hamster was smaller than that of diabetic hamster. In vivo experiments of hypoglycaemia supported the in vitro experiments. When given buccally, bacitracin, aprotinin and sodium deoxycholate could increase the relative pharmacological bioavailability of insulin. When co administered with aprotinin (0.1%), bacitracin (0.5%) and sodium deoxycholate (1%), the relative pharmacological bioavailabilities of insulin were 4.84%, 7.52% and 9.60%, respectively. CONCLUSION The in vitro and in vivo results suggest that proteolytic enzymes limit absorption of insulin in the buccal. The enzyme activity should be minimized in order to improve the bioavailability of insulin via buccal delivery.