Abstract: AIM　To study the bioavailabilities of insulin (INS) aerosol given by pulmonary route to rats. METHODS　Two-phase INS aerosol in propellant system was prepared and the amount of insulin delivered per actuation was determined by HPLC method. INS aerosol was delivered by oral inhalation or intratracheal administration to rats. Blood glucose levels and serum INS concentrations were examined by glucose oxidase method and radioimmunoassay, respectively. The relative bioavailabilities were then calculated. RESULTS　By HPLC determination, the amount of insulin per actuation of two different doses of INS aerosol was 0.39 u for low dose and 1.45 u for high dose. When delivered to rats, INS aerosol significantly reduced blood glucose level and increased serum INS concentrations by inhalation as well as intratracheal delivery at low or high doses. The relative bioavailability of the drug reached 24.3% by inhalation and 25.7% by intratracheal delivery at low dose. CONCLUSION　Results indicated that INS aerosol showed higher bioavailability when given pulmonarily to rats.