The structural factors of antimycin A molecule required for inhibitory action were studied using newly synthesized antimycin A derivatives with bovine heart submitochondrial particles, in order to probe the interaction between antimycin A and its binding site. In particular, we focused upon the roles of the amide bond bridge, which connects the salicylic acid and dilactone ring moieties, and the 3-formylamino group in the salicylic acid moiety. The lack of formation of an intramolecular hydrogen-bond between phenolic OH and amide carbonyl groups resulted in a remarkable loss of the activity (by four orders of magnitude), indicating that this hydrogen-bond is essential for the inhibition. This result suggested that both the phenolic OH and the carbonyl groups form a hydrogen-bond with some residues at a fixed conformation. In addition, the inhibitory potency was remarkably decreased by N-methylation of the amide bond moiety, indicating that the NH group might function in hydrogen-bond interaction with the binding site. The N-methylation of 3-formylamino group also resulted in a decrease in the activity, probably due to a loss of the rotational freedom of this functional group. Molecular orbital calculation studies with respect to the conformation of the 3-formylamino group indicated that this group takes an active conformation when the formyl carbonyl projects to the opposite side of the phenolic OH group. Based upon a series of structure-activity studies of synthetic antimycin A analogues, we propose a tentative model for antimycin A binding in its binding cavity.