Pyrrolosporin A (1) is a new macrolide antitumor antibiotic possessing an unusual spiro-alpha-acyltetronic acid moiety. The antibiotic was isolated from the fermentation broth of Micromonospora sp. by vacuum liquid chromatography, crystallization and reversed phase HPLC (C18). The structure was determined by a combination of NMR, MS, IR, UV, X-ray analysis and degradation studies.