Myxobacteria are proficient producers of biologically active secondary metabolites. However, efforts to exploit these natural products for the development of new therapeutics and agrochemicals are frequently hampered by low production levels. We describe here a transposon-based strategy to identify genes encoding regulators of secondary metabolite biosynthesis in the myxobacterium Angiococcus disciformis An d48, which produces the highly efficient electron transport inhibitor myxothiazol. Extracts from 1200 transposon mutants were analyzed by HPLC, leading to the identification of six mutants in which myxothiazol production was increased by as much as 30-fold. Identifying the sites of integration coupled with sequencing of flanking regions, showed that some of the inactivated genes encode proteins with similarity to known bacterial regulators such as two-component systems and serine-threonine protein kinases. However, other gene products do not resemble any characterized proteins. Taken together, these data show that this transposon-based strategy is a valuable tool to identify regulatory genes of secondary metabolism, including gene loci which cannot be detected using current in silico approaches.