Sisomicin, the principal antibiotic produced in the fermentation of Micromonospora inyoensis, has been shown to be O-2,6-diamino-2,3,4,6-tetradeoxy-α-D-glycero-hex-4-enopyranosyl(1→4)-O-[3-deoxy-4-C-methyl-3-(methylamino)-β-L-arabinopyranosyl(1→6)]-2-deoxy-D-streptamine (1). Sisomicin contains a novel unsaturated sugar unit, not previously encountered in any aminocyclitol antibiotic. Submerged fermentations of Micromonospora inyoensis (NRRL 3292) produce sisomicin, a novel unsaturated aminocyclitol antibiotic having broad spectrum antibacterial activity. Sisomicin is the major component of the crude antibiotic complex, which was isolated from the fermentation broth by ion-exchange chromatography. Column chromatography of the crude antibiotic on silica gel afforded pure sisomicin, which was crystallized from ethanol. Chemical and physical studies have established structure 1 for sisomicin.