Cyclosporin A is synthesized by cyclosporin synthetase, a multienzyme polypeptide. This enzyme catalyzes at least 40 reaction steps in an assembly belt-like mechanism. It activates all constituent amino acids of cyclosporin A to thioesters via amino acyladenylates and carries out specific N-methylation reactions. During elongation, the activated amino acids are linked by peptide bonds leading to enzyme-bound nascent peptide chains. Some of the linear peptides of the growing cyclosporin A chain were isolated and their N-terminal amino acid was determined. D-Alanine at position 8 of the cyclosporin A molecule was found to be a starting amino acid in the biosynthetic process of cyclosporin A formation. Four intermediate peptides of the growing peptide chain of cyclosporin A could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. That these intermediate peptides were bound by thioester linkage to cyclosporin synthetase could be demonstrated by liberation of the peptides with performic acid. The peptides strongly suggest the stepwise synthesis of a single linear peptide precursor of cyclosporin A.