Tunicamycin (I) is a nucleoside antibiotic complex produced by Streptomyces lysosuperficus. In the preceding communication, the structure of its degradation product, tunicaminyl uracil (II), was reported. This paper deals with the isolation of other degradation products and the gross structure of tunicamycin. Tunicamycin was hydrolyzed with 3 N hydrochloric acid, and the hydrolyzate was extracted with ethyl ether to obtain a mixture of fatty acids (III). IR, NMR, GC-MS, hydrogenation, oxidation, chromatography, and other analyses showed that tunicamycin is a mixture of homologous antibiotics, each containing one mole of fatty acid with different carbon chain lengths; the major fatty acids are trans α,β-unsaturated iso acids, such as 13-methyl-2-tetradecenoic acid (III A), 14-methyl-2-pentadecenoic acid (III B), 12-methyl-2-tridecenoic acid (III C), and 15-methyl-2-hexadecenoic acid (III D), which correspond to tunicamycin A, B, C, and D respectively. Hydrolysis of tunicamycin also yielded D-glucosamine hydrochloride. Through 1H NMR, specific rotation, IR spectrum, and mass spectrometry analysis of its permethylated derivative (V), it was suggested that the C-11' of the tunicamine residue is linked to the C-1" of N-acetyl glucosamine via an α-α glycosidic linkage. The structure I was assigned to the main components of the tunicamycin complex.