Abstract-The electron-impact induced fragmentation of staphylomycin S (I) and related cyclodepsipeptides (I1 to V) is mainly initiated by elimination of an azomethin molecule (R-CH=N-R), i.e. 1,2-dehydro-4-oxopiperidine for I and 11, 1,2-dehydr0-4-hydroxy-piperidine for 111 and IV, and an unknown hydroxy-l,2-dehydro-4-oxopiperidiiie for VI. Cyclic imino acids, e.g. a pipecolic acid derivative for I to IV and 4-hydroxyproline for etamycin (IX) preferentially trigger this reaction. Clearcut evidence for the amino acid sequence is given by the mass spectra of the linear peptides obtained from I, V and IX by alkaline hydrolysis of the lactone function and esterification of the carboxy group. THE ISOLATION and structure determination of staphyloniycin S (I), one of the components of the antibiotic staphylomycin, has been described ear1ier.l The identification of the amino acids and oligopeptides, obtained after total and partial hydrolysis, provided the structural features of this compound. The increasing importance that mass spectrometry has won in recent years in establishing the sequence of amino acids in peptides indicated an alternative means of reinvestigating the structure of staphylomycin S. Furthermore some naturally occurring modifications, both known (11) and unknown (V, VI) and two epimeric dihydro derivatives (111, IV) were available to assist in (i) assigning possible structures to the fragment ions and (ii) determining the utility of these fragmentations for structure elucidation of peptides related to I.