The biosynthesis of small-size polyene macrolides is ultimately con-trolled by acouple of transcriptional regulators that act in a hierarchical way. AStreptomycesantibiotic regulatory protein–large ATP-binding regulator of the LuxRfamily (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encodinggene and activates its transcription, and in turn, the gene product of the latter acti-vates transcription from various promoters of the polyene gene cluster directly. Theprimary operator of PimR, the archetype of SARP-LAL regulators, contains three hep-tameric directrepeats separated by four-nucleotide spacers, but the regulator canalso bind a secondary operator with only two direct repeats separated by a3-nucleotide spacer, both located in the promoter region of its unique target gene,pimM. A similar arrangement of operators has been identified for PimR counterpartsencoded by gene clusters for different antifungal secondary metabolites, includingnot only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cyclohex-imide. Here, we used promoter engineering and quantitative transcriptional analysesto determine the contributions of the different heptameric repeats to transcriptionalactivation and final polyene production. Optimized promoters have thus been devel-oped. Deletion studies and electrophoretic mobility assays were used for the defini-tion of DNA-binding boxes formed by 22-nucleotide sequences comprising twoconserved heptameric direct repeats separated by four-nucleotide less conservedspacers. The cooperative binding of PimRSARPappears to be the mechanism involvedin the binding of regulator monomers to operators, and at least two protein mono-mers are required for efficient binding.