A sisomicin fermentation carried out in the presence of (methyl-¹⁴C)-L-methionine resulted in a crude mixture, composed of methyl-¹⁴C-labeled sisomicin as a major component; and two 4"-C-desmethylsisomicin (66-40B and 66-40D) isomer-like components, an unidentified component and a gentamicin A-like antibiotic as minor components. When (methyl-¹⁴C)-L-methionine was added in an early stage of the fermentation (24 hours), incorporation of methyl-¹⁴C-label into polar components (e.g., gentamicin A-like antibiotic) preceded that into sisomicin. Chromatographic evidence for the bioconversion of (methyl-¹⁴C)-gentamicin A to a radioactive sisomicin-like product (possibly (3"-N-methyl-¹⁴C)-sisomicin) was seen, when a Micromonospora blocked mutant was incubated in the presence of the former antibiotic.