In a recent report, the structure originally advanced for the nucleoside antibiotic, gougerotin, (I) was revised to structure II on the basis of chemical and physical evidence. The establishment of the carbohydrate moiety as a 4-aminohexose derivative and the positional assignment of the peptide to the 4'-amino group was determined by chemical studies. The elucidation of the configuration at C-1' as beta and C-5' had already been shown by Iwasaki. By nmr spectroscopy of III (prepared from gougerotin), we established the configuration at C-2' and C-3'. These studies left only two possibilities for the identity of the sugar moiety of this antibiotic, namely, the gluco or galacto configuration. Iwasaki had isolated from the degradation of gougerotin colorless needles of a methyl aminohexoside tetraacetate (IV), mp 193', [α]D +87', to which he assigned structure IV. On the basis of our studies on derivative III, we revised structure IV to V, an α or β derivative of a methyl 4-amino-4-deoxy-glucoside or -galactoside. Since the physical properties of V differed from the reported values of the α and β derivatives of isomeric methyl 4-amino glucosides (VI and VII), we assigned the galacto configuration (II) to gougerotin and structure VIII to the carbohydrate derivative IV isolated by Iwasaki. Our data did not permit assignment of the anomeric configuration to the carbohydrate moiety of VIII. We prepared compound V from gougerotin: Treatment of III with sodium borohydride in 5% aqueous methanol gave the 5'-hydroxymethyl derivative (IX) in 8% yield, mp 245° (dec), which was hydrogenated over platinum oxide to afford the trimethyleneurea nucleoside X, mp 270-271° (dec), in quantitative yield. Methanolysis of X followed by acetylation in pyridine gave colorless needles (V) in 3% overall yield from IX with mp 193.5-194° and [α]D +75°. However, the nmr spectrum of V showed two signals of equal intensity for the methoxyl protons at δ = 3.38 and 3.47 indicating a mixture of anomers. Thick layer chromatography (2 mm) on Silica Gel PF254 using benzene-MeOH (10:1) by multi-development technique gave a partial separation (visualized by ferric hydroxamate reagent) in the form of an elongated band. Removal of the upper portion of this band followed by extraction with a 1:1 mixture of acetone-CHCl3 and evaporation of the solvent gave a syrup which crystallized from isopropanol, mp 151-153°. Further recrystallization to constant mp 140-146° gave a material with [α]D +146' (c, 0.58 in CHCl3) suggesting the α-gluco structure VI. Conclusive proof of the α-gluco configuration was obtained from nmr (including decoupling experiments) and IR spectroscopy. From the lower portion of the elongated band in the thick layer chromatogram, the essentially pure beta anomer VII was obtained by a similar isolation procedure, mp 195.5-196°, [α]D +5° (c, 0.8 in CHCl3) which compares favorably with the constants reported for the β-gluco isomer VII by von Salta et al. From these data, we conclude that V obtained from gougerotin is a mixture of the gluco anomers VI and VII and that III has the gluco configuration. Consequently the structure of gougerotin is 1-(cytosinyl)-4-sarcosyl-3-deoxy-1,4-dideoxy-β-D-glucopyranuronamide (XI).