Alisamycin is a new manumycin group antibiotic. Manumycin group antibiotics have three asymmetric carbons at C4, C5, and C6 in their structures. The absolute structure of alisamycin was determined by the exciton chirality method and CD spectrum of the oxidative product of alisamycin. Configurations of C4, C5, and C6 were assigned to be 4R, 5S, and 6R.