A simple method has been developed for the characterization of neutral polyene antibiotics, involving conversion of the antibiotic to its polyacetate in acetic anhydride-pyridine. Mass spectra of these derivatives contain abundant molecular ions and the number of hydroxyl groups in the antibiotic can be inferred from the number of moles of acetic acid lost. This method is often superior to trimethylsilylation since the derivatives themselves are stable to hydrolysis and give good molecular ions. Application of this method to filipins I, II, III and IV assigned them molecular formulas C35H58O9, C35H58O10, C35H58O11, and C35H58O11, respectively. The number of hydroxyl groups was determined to be 7, 8, 9, and 9, and the number of double bonds was 5 for all components. Filipin I has two hydroxyl groups fewer than filipin III, filipin II has one hydroxyl fewer than III, and filipin IV is apparently isomeric with filipin III. These conclusions agree well with the available chromatographic data, where filipins III and IV (with more hydroxyl groups) move slower than the others on both thin-layer and partition chromatographies.