The final assembly of the 11-peptide chain of cyclosporins and its cyclization is accomplished in the producer Beauveria nivea by cyclosporine synthetase. This multienzyme represents the largest integrated enzyme structure reported so far. Its size has been estimated to approximately 1,500 kDa by SDS-PAGE. Some enzyme bound linear peptides could be isolated and identified. All of them represent partial sequences of cyclosporin A starting with D-alanine. These peptides were bound by thioester linkage to the enzyme. This could be demonstrated by liberation of the peptides with performic acid and by inhibition of in vitro cyclosporin A synthesis with thiol blocking agents. Cyclosporin synthetase is capable to synthesize besides a lot of cyclosporins known from fermentation studies some new cyclosporins so far not obtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, Dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. We were able to synthesize these cyclosporins in sufficient quantities to ensure their structure by fast atom bomdardment mass spectroscopy and to examine their immunosuppressitivity. All new cyclosporins synthesized in the in vitro system so far are immunosuppressive.