Two putative ABC transporter genes pdmR1 and pdmR2 are found in the biosynthetic gene cluster of pradimicin. pdmR1 showed a hydrophilic profile with similarity to the daunorubicin-resistant ABC transporter ATP-binding protein family, a characteristic ABC transporter. pdmR2 codes for hydrophobic polypeptides showing a high degree of similarity to several ABC-type multidrug transport systems. To assess the possible roles of these genes in pradimicin biosynthesis, pdmR1 and pdmR2 were introduced into Actinomadura hibisca P-1752. The recombinant strains Actinomadura hibisca SP1, SP2, and SP3 showed longer life spans compared to the wild-type A. hibisca. Pradimicin production was increased by 3.5, 2.4, and 5.4-fold at 8 days in A. hibisca SP1, SP2 and SP3, respectively, compared to that of parental strain. The higher transcriptional level of pdmR1 and pdmR2 genes in the pdmR1 and pdmR2 harboring strains compared to the wild type was consistent with the enhanced production. The plasmids pSP1, pSP2, and pSP3 were expressed in Streptomyces venezulae and S. lividians, which are sensitive to pradimicin. The transformants acquired higher resistance to pradimicin than the wild-type Streptomyces strains suggesting that pdmR1 and pdmR2 acts as a set of resistance genes in A. hibisca to pump out pradimicin produced throughout the cell.