Apramycin, C21H41N5O11, a broad-spectrum aminocyclitol antibiotic, is produced by a strain of Streptomyces tenebrarius. It does not fall into the tobramycin, kanamycin, gentamicin group and a detailed examination of its structure involving degradative, synthetic, and spectroscopic analysis leads to the assignment of an unusual structure, the main features of which are a 4-amino-4-deoxy-D-glucose moiety, a 1-1' sugar linkage, and an octadiose which exists as a rigid bicyclic system. The proposed structure is confirmed by an x-ray diffraction study.