Geldanamycin (Gdm), an inhibitor of heat shock protein 90 (Hsp90), shows antitumor and antivirus bioactivity. Most Geldanamycin biosynthetic genes have been cloned from the genome library of Streptomyces hygroscopicus 17997. In this report, polyketide synthase (pks) gene, mono-oxygenase (gdmM) gene and carbamoyltransferase gene (gdmN) were subjected to inactivation. Three gene disrupted mutants (△pks, △gdmM and △gdmN) were obtained by double crossover. No Geldanamycin production was detected in three mutant strains cultured in fermentation broth. Gene complementation experiments excluded the possible polar effect of gene disruption on other genes. These results confirmed that pks, gdmM and gdmN genes were essential for Geldanamycin biosynthesis.