Cycloheptamycin, an antibiotic from an unidentified Streptomyces species, was examined by mass spectrometry after N-permethylation The structure was thereby determined as a cyclic derivative of HCO.VaI.Thr.-Tyr(Me)Ala.MurIle.&hydroxyNva.5-methoxyMeTrp.O~ in which a lactone is formed between the C-terminal acid and the threoninc OH function. N-methylalloisoleucine and f&hydroxynorvaline were isolated and identified by comparison with authentic compounds and the contiguration of several of the amino acids was determined enzymatically.