An inosamine-idiotrophic mutant, KY11559, which produced no astromicin unless scyllo-inosamine was added to the fermentation medium, was isolated from Micromonospora olivasterospora. Biotransformation studies were performed with resting cells of this mutant and compounds assumed to be precursors of 1,4-diaminocyclitol (fortamine). Scyllo-inosose, scyllo-inosamine and FU-10 were converted to astromicin. A number of mutants blocked in the biosynthesis of astromicin were developed from M. olivasterospora, and the intermediates accumulated by these mutants were isolated and identified. Twenty-five blocked mutants were classified into 10 groups, based on their complementation patterns by cosynthesis experiments. Further, utilizing these blocked mutants and the isolated compounds, biotransformation analyses were performed. The results showed that the amination at position 4 in fortamine occurred after formation of the pseudodisaccharide. Subsequently, the aminosugar and aminocyclitol moieties were aminated, methylated, dehydroxylated, epimerized and acylated to produce astromicin. Thus it was demonstrated that the astromicin biosynthetic pathway has a unique feature which is not found in the biosynthesis of other aminoglycoside antibiotics.