The response regulator MtrA is highly conserved in Streptomyces species where it coordinates antibiotic production with sporulation. Loss of MtrA results in a conditional bald phenotype in S. avermitilis, S. coelicolor and S. venezuelae, where the production of aerial hyphae and spores is blocked on some but not all growth media. In S. venezuelae NRRL-B-65442 ChIP-seq showed that MtrA binds to the promoters of >1000 genes including key genes required for development. Here we show that an S. venezuelae NRRL-B-65442 ∆mtrA mutant is bald on R2YE agar but develops normally on MYM agar. We used an in vitro DNA footprinting technique called Reusable DNA Capture Technology combined with Surface Plasmon Resonance (ReDCaT SPR) to identify the precise MtrA binding sites at the promoters of the developmental genes adpA, bldM, dnaA, filP, mtrA, ssgB and whiI. The results show that purified MtrA binds to single sites upstream of filP, mtrA, ssgB and whiI and to multiple sites upstream of adpA (x2), bldM (x5) and dnaA (x3) which may be indicative of the complex regulation of these genes. Tandem mass tag proteomics on the wild-type and ∆mtrA strains revealed that the levels of BldM and WhiI are 4-fold and 7-fold lower in the ∆mtrA mutant grown on R2YE agar suggesting they are directly regulated by MtrA under these growth conditions. They were not significantly affected in cultures grown on MYM agar. The expression of the key BldM and WhiI target genes ssgB, ssgR and whiB, which are key to development is also significantly reduced on R2YE agar. We conclude that MtrA directly activates expression of bldM and whiI on R2YE agar and that reduction in the levels of these regulators leads to the bald phenotype of the ∆mtrA mutant on this growth medium.