The rubradirin biosynthetic gene cluster harbors 58 ORFs within a 105.6-kb sequence, which includes all of the genes responsible for the synthesis of rubradirin, as well as the primary genes relevant to regulatory, resistance, and transport functions. This gene cluster also harbors a resistance-mediating ABC transporter, RubT1, which is located at the most upstream position in the cluster. In the present study, RubT1 was expressed heterologously in E. coli, and the resistance affinity of RubT1 was determined by an antibacterial activity test, as well as by HPLC and ESI-MS analyses. Evidence clearly demonstrates that RubTl mediates rubradirin resistance as an ABC transporter.