Antibiotic nystatin, produced by Streptomyces noursei, is a complex containing three biologically active components (A1, A2, A3), all of which are polyene macrolides with a diene-tetraene chromophore and glycosidically linked mycosamine moiety. The structure of the main component A1 has been determined. This paper reports the complete structure of nystatin A3 following preliminary studies. Nystatin A3 was isolated from commercial nystatin via a two-step countercurrent chromatography (CCD) method: first, A1 was separated using a chloroform-methanol-1% aqueous NaCl (2:2:1) system after 400 transfers; then, the residue was separated into A2 and A3 using a chloroform-methanol-borate buffer pH 8.2 (2:2:1) system after 400 transfers. Physicochemical properties of nystatin A3: colorless amorphous powder, insoluble in water, soluble in alcohols, DMF, DMSO, and pyridine; UV spectrum (methanol) with maxima at 290, 304 (E1%m 650), and 318 nm; fast atom bombardment mass spectrum (FAB-MS) showed m/z 1056 (M+H)+. The structure of nystatin A3 (I) was elucidated by spectroscopic analysis of its degradation products using the procedure previously applied to nystatin A1. The only structural difference between A1 and A3 is the presence of an L-digitoxose moiety in A3. Evidence for the location of L-digitoxose: ozonolysis of A3, followed by hydrogenation of ozonides, reduction with sodium borohydride/sodium borodeuteride, and hydrolysis with barium hydroxide yielded 3-(O-digitoxosyl-1)-2,4-dimethylhexane-1,5-diol (II) and its 1-monodeuterio analogue (III). These products were converted to tetra-O-methyl derivatives (IV, V) and analyzed by MS. Methanolysis of IV produced 1,5-di-O-methyl-2,4-dimethylhexane-3-ol and tri-O-methyl-L-digitoxose (identified by MS and 1H NMR), indicating L-digitoxose is glycosidically bound at C-35 of A3. The remaining structure of A3 was assigned by MS analysis of degradation products from a sequence of reactions (ozonolysis, hydrogenation, reduction, oxidation, hydrolysis), which yielded polyols (e.g., 2,4-dimethylhexane-1,3,5-triol, decane-1,3,5,7,10-pentaol) identical to those from A1. All results enabled the postulation of nystatin A3's structure, and it is proposed to form a hemiketal bond involving C-13 and C-17, analogous to other diene-tetraene polyene macrolides.