The mechanism of action of peptide inhibitors on angiotensin I converting enzyme (AI converting enzyme) was studied in relation to the substrate requirements of the enzyme in vivo in the dog lung and in vitro in plasma. 1 d Asp 8 Ile AI prepared by the solid phase technique was compared with the known peptide inhibitors of AI converting enzyme, BPP(5a) (Pyr Lys Trp Ala Pro) and SQ 20881 (Pyr Trp Pro Arg Pro Gln Ile Pro Pro). 125I 1 d Asp 8 Ile AI was evaluated for susceptibility to cleavage by the AI converting enzyme. In vitro and in vivo, 125I 1 d Asp 8 Ile AI or BPP(5a) in 50,000 fold molar excess produced only a slight delay in conversion of 125I AI to 125I angiotension II (AII); SQ 20881 blocked conversion completely. In vivo, 1 d Asp 8 Ile AI or BPP(5a) injected into the pulmonary circulation in 250 fold molar excess (250 nmole/kg) did not cause a dimunition in the pressor response to AI administered 30 sec. later; SQ 20881 blocked the pressor response to AI for 60-90 min. 1 d Asp 8 Ile AI was a poor substrate for converting enzyme, since 125I 1 d Asp 8 Ile AI was not converted to 125I 1 d Asp 8 Ile AII and unlabelled 1 d Asp 8 Ile AI did not block the pressor response to exogenous AII. 125I 1 d Asp 8 Ile AI was stable in plasma and in the pulmonary circulation. The results suggest that since 1 d Asp 8 Ile AI is neither a substrate nor a blocker, substitution of the aliphatic residue Ile in the 8 position of AI may prevent binding to an active site in the AI converting enzyme.