Two polyene antibiotics, nystatin and amphotericin A, were compared by physico-chemical and microbiological methods. The two antibiotics were found to have the same molecular weight, 926, by plasma desorption and electron-impact MS. However, 13C NMR spectrometry and HPLC studies indicated that the two molecules are different. The 200 MHz NMR studies indicated a chemical environment of 24 carbons of amphotericin A identical with that of the carbons of amphotericin B and nystatin. The structure of amphotericin A is identical with that of amphotericin B, except that there is a single bond between carbons 28 and 29 instead of a double bond, as shown by two-dimensional NMR studies.