Endothelin converting enzyme (ECE) is essential for generation of the biological effects of endothelin-1 (ET-1) from a precursor, big endothelin-1 (Big ET-1). We synthesized four analogs of human Big ET-1[16-38], substituted with single D-amino acids at P1, P2, P1' and P2' positions. ECE activity was determined using an ET-1 specific radioimmunoassay system. None of the D-amino acid containing Big ET-1 analogs were apparently cleaved by ECE, however, one of the synthetic peptides, [D-Val22]Big ET-1[16-38], strongly inhibited the ECE activity. Furthermore, when this D-Val22 containing peptide was preadministered to rat striatum, it was found to inhibit the dopamine release induced by Big ET-1. This result suggests that the D-Val22 containing peptide inhibits the ECE activity in vivo. The D-Val22 containing inhibitor offers hope of developing more potent and highly specific ECE inhibitors of therapeutic significance.