Nystatin, the most commonly used clinical polyene macrolide antifungal antibiotic from Streptomyces noursei and first discovered member of this group of natural products, has a rather long record of chemical studies. It was generally characterised and the structure of glycosidic constituent (mycosamine) elucidated. Extensive chemical studies led to a rather deep insight into the structure of the antibiotic, and in the most advanced studies the structure of aglycone (nystatinolide) was postulated, although the place of attachment of mycosamine moiety still remained unknown. Recent finding that nystatin is not an individual compound but a mixture of two active principles (both tetraenes), named nystatin A1 and A2, required the verification of accumulated structural data as based on the use of unresolved antibiotic complex. In the present report the complete structure (I) of nystatin A1 (main component of the complex) is postulated. The pure component was isolated from the complex according to the previously described method. The reaction sequence (f) led to the formation of compound (IX) which on mass spectrometry exhibited M+ at m/e 296 and fragment ions (XVII), thus the carbon skeleton of C1_20 fragment of (I) is that of 5-methyl eicosane. Degradations (c,d,e) allowed the localisation of functional groups in C1_10 fragment of (I). Decapenta-ol (1,3,5,7,10) (VII) was examined by mass spectrometry in form of pentamethyl ether derivative (XX), found M+ at m/s 292 and fragment ions (XVIII); C1 of (VII) was derived from carboxyl and C10 from aldehydo (formed in periodate cleavage of C9-C10 vic. glycol in (I)) because selective deuterisation in reactions (e) led to the formation of 1,1,10-trideutero analogue (VIII) of (VII), pentamethyl ether of (VIII) gave M+ at m/e 295 and fragment ions (XIX) on mass spectrometry. Functional groups in C11_20 fragment of (I) were localised in reactions (g,h,i,j,k); the basic compound was 6-hydroxymethyl-decapentaol (1,3,5,7,10) (XI) formed in reactions (h,j), its heptamethyl ether gave M+-31 at m/e 335 and fragment ions (XX) on mass spectrometry; hydroxymethyl at C6 of (XI) was derived from carboxyl group at C16 of (I) as demonstrated by selective introduction of two deuterium atoms in (XIII) in reactions (k), heptamethyl ether of (XIII) exhibited M+-31 at m/e 337 and fragment ions (XXIII); hydroxyl groups at C3 and C10 of (XI) were derived from ketone group and chromophore terminus at C13 and C20 of (I) respectively as shown by selective deuterisation in procedures (h,j), heptamethyl ether of (XII) gave M+-31 at m/e 337 and fragment ions (XXII); finally C1 hydroxymethyl of (XI) was derived from C11 vic. glycol hydroxyl of (I) to be in accordance with structure of (IX); oxygen function at C18 of (I) is hydroxyl as ketone compound (XII) would contain a third deuterium atom at C18. Mycosamine moiety is attached glycosidically at allylic C19 of (I), which is in accord with extreme ease of acid hydrolysis of glycosidic bond and elimination of mycosamine but not methyl mycosaminide in MeOH/HCl; the suggested reaction mechanism is indicated on Fig.2; elimination reaction with good yield leads to formation of new conjugated double bond, transformation of light absorption spectrum of (I) from tetraene to pentaene is shown on Fig.1, formation of pentaene points to chromophore terminus at C20 of (I) belonging to tetraene not diene conjugated system (the latter also present in antibiotic molecule). Ring structure of mycosamine moiety is pyranose type; mild hydrolysis of peracetyl derivative of (I) (reactions l) yielded 2,3,4-triacetyl mycosamine (XIV), its pyranose ring structure and C-1 conformation were based on determination of chemical shift and spin-spin coupling constants in 80-MHz NMR (CDCl3). Structure of carbon skeleton of tetraene and diene chromophores portion including C19_35 fragment of (I) was established in reactions (a); dimethyl ester of 2-methyl-heptadecanedioic acid obtained (II) exhibited mass spectrum identical with synthetic product; tetraene and diene chromophores are separated by -CH2-CH2- fragment which gives rise to 1,4-dideutero-butanediol (1,4) (III) formed in reactions (b), TMS derivative of (III) had retention time in GC identical with authentic sample, deuterium atoms in (III) were localised by mass spectrometry of TMS derivative. Structure of remaining fragment of (I) including C33_38 was established in reactions (b,c,d); compound (VI) obtained in reactions (c,d) gave M+ at m/e 204 and fragment ions (XV) on mass spectrometry of its trimethyl ether; attachment of this moiety to terminus of diene chromophore in (I) was established by selective deuterisation in reactions (b), monodeutero analogue (IV) of (VI) exhibited M+ at m/e 205 and fragment ions (XVI) on mass spectrometry of its trimethyl ether derivative, introduction of only one deuterium atom proves remaining oxygen functions in corresponding moiety of (I) are hydroxyls not ketone. NMR examination of compound (V) formed in reactions (c) supplied direct evidence for position of lactone bond between C1 and C37 of (I); multiplet signal centered at 5.3 ppm (d5-pyridine:CDCl3=1:5) was assigned to most unshielded proton at carbon atom with acyloxy group, which is C5 proton of (V) because irradiation with its resonance frequency caused transformation of 1.05 ppm doublet (corresponding to most unshielded CH3 at C5 of (V)). The postulated complete structure of nystatin A1 shows striking similarities with heptaene macrolide antibiotic amphotericin B.