The mutants blocked in the gentamicin C₁ production were derived from a sagamicin producing strain of Micromonospora sagamiensis. The intermediates produced by these mutants were isolated and properly identified. Comparing the biotransformation activities in the resting cells of the mutants with those of a DOS idiotroph, KY 11525, the blocked steps in sagamicin and gentamicin biosynthesis were proposed in each mutant. Mutant KY 11564 was found to produce gentamicin C_2a (C-6'-epimer of gentamicin C₂) together with gentamicin C_1a, and sagamicin. KY 11525 transformed gentamicin C_2a into C₂ and C₁, whereas KY 11564 lacked the activity. KY 11565 produced gentamicin X₂ and antibiotic G-418, and lacked 6'-amino substitution activities. KY 11566 appeared to be partially blocked in 6'-N-methylation activities, and the major products were gentamicin C_2a and C_1a. From these results, sagamicin biosynthesis in M. sagamiensis is discussed.