5-Deoxygentamicin C.sub.2b and 6'-N-methyl-2-hydroxy- and 6'-N-methyl-5-deoxygentamicins C.sub.2b are prepared, as the major fermentation products, by culturing a nutrient medium containing carbohydrates, a source of assimilable nitrogen, essential salts and either D-streptamine or 2,5-dideoxystreptamine with a mutant of Micromonospora purpurea and isolating the fermentation products from the nutrient medium. The fermentation products are acylated with an ester of an .omega.-[N-(benzyloxycarbonyl)amino]-.alpha.-hydroxy-lower-alkanoic acid after first blocking the primary amino group at the 2'-position with an amine-protecting group, followed by catalytic hydrogenolysis of the benzyloxycarbonyl group and removal of the amine-protecting group to prepare the 1-(.omega.-amino-.alpha.-hydroxy-lower-alkanoyl) derivatives.