Abstract: In order to verify the function of genD1, a recombinant plasmid pGD14 was constructed for blocking-up genD1. pGD14 was introduced into Micromonospora purpurea GK1101(ΔgenK))by conjugation. A desired mutant strain GD1989 was obtained by PCR analysis. Flask shaking experiments and MS analysis showed that the mutant strain GD1989 only produced gentamicin A instead of gentamicin C complex, and the product of GD1989 feeding on gentamicin X2 contained gentamicin C complex. It was speculated that genD1 gene might be responsible for methylation at C-4″ of garosamine.