A technique previously used to isolate a mutant of Streptomyces fradiae capable of synthesizing the antibiotic neomycin only in the presence of the subunit 2-deoxystreptamine has been applied to S. rimosus forma paromomycinus and S. kanamyceticus to produce mutants capable of synthesizing paromomycin and kanamycin, respectively, only in the presence of added 2-deoxystreptamine subunit. Neamine, paromamine, and 6-kanosaminido-2-deoxystreptamine—2-deoxystreptamine-containing glycosides that are plausible intermediates in the biosynthesis of the three antibiotics—were also tested as substrates. The mutants studied did not convert the glycosides to active antibiotics. The mutant of 5. rimosus forma paromomycinus converts streptamine, an analog of 2-deoxystreptamine, to two new antibiotics, hybrimycins C1 and C2, analogs of paromomycins I and II, respectively, in which the only modification is that 2-deoxystreptamine has been replaced by streptamine. Selective hydrolysis yielded a third new antibiotic, hybrimycin C3, an analog of paromamine.