By using 2 neamine-negative mutants, MCRL 5003 and MCRL 5004, of Bacillus circulars, subunit assembly in the biosynthesis of butirosins was investigated. The two mutants were blocked at different steps in the biosynthetic pathway of butirosins, but could produce butirosins when the culture medium was supplemented with neamine. Mutant MCRL 5003 accumulated 2-deoxystreptamine (DOS) in the fermentation broth but could not utilize DOS for the biosynthesis of butirosins, whereas mutant MCRL 5004 could produce butirosins from DOS. Based upon the ability of these 2 mutants to incorporate a series of DOS-containing compounds considered as plausible biosynthetic intermediates of butirosins into butirosins or related antibiotics, the biosynthetic pathways for butirosins and 6'-deamino-6'-hydroxybutirosins were proposed. Feeding experiments with 3', 4'-dideoxy derivatives of neamine, ribostamycin and butirosins supported the pathway proposed for butirosins. A glucosamine auxotroph MCRL 5771 of B. circulars afforded some information as to the role of D-glucosamine in the biosynthesis of butirosins.