The structure of the potent antifungal antibiotic scopafungin has been assigned as 1, featuring a 36-membered macrolide ring, an unsymmetrically disubstituted guanidine, and a monoester of malonic acid. The molecular formula assigned, C59H103N3018r agrees with high-resolution fast atom bombardment and field desorption mass spectrometric measurements. The structure of scopafungin was derived by combining structures assigned to key ozonolysis and periodate products, based on 'H (including two-dimensional FT) and I3C NMR spectroscopy and on high-resolution field desorption and electron ionization mass spectrometry. All alkene linkages were assigned as trans from coupling constants and examination of the two-dimensional 'H NMR spectrum, while relative stereochemistry at C-6 through C-9 and C-25 through (2-28 was assigned from coupling constants. Extensive use of reversed-phase high-performance liquid chromatography was made in separating the complex mixtures of scopafungin components and degradation products.