The effects of absorption enhancers and protease inhibitors on the pulmonary absorption of insulin and (Asu 1 , 7 )eel-calcitonin (ECT) were examined by means of an in vivo pulmonary absorption experiment. Absorption enhancers used in this study were sodium-glycocholate (Na-GC), linoleic acid-surfactant mixed micelles (MM), N-lauryl-β-d-maltopyranoside (LM), ethylenediamine tetraacetic acid disodium salts (EDTA), sodium caprate (Na-Cap) and sodium salicylate (Na-Sal) whereas aprotinin, bacitracin, soybean trypsin inhibitor (STI) were used as protease inhibitors. The absorption of insulin and ECT from the lung was evaluated by their hypoglycemic and hypocalcemic effects, respectively. We found significant and continuous hypoglycemic or hypocalcemic effects after the insulin or ECT administration with these additives. Of these adjuvants, 10 mM LM appeared to be more effective for enhancing the pulmonary absorption of these peptides than the same concentration of other adjuvants. In addition, the protease inhibitors were effective in enhancing the pulmonary absorption of insulin due to their inhibitory action of the insulin degradation. Furthermore, these adjuvants did not cause severe mucosal damage of rat lung, as determined by the leakage of Evans blue from the systemic circulation. These findings suggest that the use of absorption enhancers and protease inhibitors would be a useful approach for improving the pulmonary absorption of biologically active peptides.