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 C2a (C-6'-epimer of gentamicin C2) together with gentamicin C1a and sagamicin. KY 11525 transformed gentamicin C2a 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 C2a and C1a. From these results, sagamicin biosynthesis in M. sagamiensis is discussed.