The novel amino-glycoside antibiotics 66-40B and 66-40D produced as minor components by fermentation of Micromonospora in yoensis have been shown to be O-2,6-diamino-2,3,4,6-tetradeoxy-α-D-glycero-hex-4-enopyranosyl-(1→4)-O-[3-deoxy-3-methylamino-α-D-xylopyranosyl-(1→6)]-2-deoxy-D-streptamine and O-2,6-diamino-2,3,4,6-tetradeoxy-α-D-glycero-hex-4-enopyranosyl-(1→4)-O-[3-deoxy-3-methylamino-β-L-arabinopyranosyl-(1→6)]-2-deoxy-D-streptamine, respectively. The novel 3-deoxy-3-methylamino-β-L-arabinopyranosyl sugar unit of 66-40D has not previously been found in any amino-glycoside antibiotic. Both 66-40B and D exhibit broad spectrum antibacterial activity.