Because most antibiotics are potentially lethal to the producing organism, there must be mechanisms to ensure that the machinery responsible for export of the mature antibiotic is in place at the time of biosynthesis. Simocyclinone D8 is a potent DNA gyrase inhibitor produced byStreptomyces antibioticusTü 6040. Within the simocyclinone biosynthetic cluster are two divergently transcribed genes,simRandsimX, encoding proteins that resemble the TetR/TetA repressor-efflux pump pair that cause widespread resistance to clinically important tetracyclines. Engineered expression ofsimXfrom a strong, heterologous promoter conferred high level simocyclinone D8 resistance onStreptomyces lividans, showing thatsimXencodes a simocyclinone efflux pump. Transcription ofsimXis controlled by SimR, which directly represses thesimXandsimRpromoters by binding to two operator sites in thesimX-simRintergenic region. Simocyclinone D8 abolishes DNA binding by SimR, providing a mechanism that couples the biosynthesis of simocyclinone to its export. In addition, an intermediate in the biosynthetic pathway, simocyclinone C4, which is essentially inactive as a DNA gyrase inhibitor, also inducessimXexpressionin vivoand relievessimXrepression by SimRin vitro.