Streptomyces tsukubaensis is a well-established industrial tacrolimus producer strain, but its molecular genetics is very poorly known. This information shortage prevents the development of tailored mutants in the regulatory pathways. A region (named bul) contains several genes involved in the synthesis and control of the gamma-butyrolactone autoregulator molecules. This region contains ten genes (bulA, bulZ, bulY, bulR2, bulS2, bulR1, bulW, bluB, bulS1, bulC) including two γ-butyrolactone receptor homologues (bulR1, bulR2), two putative gamma-butyrolactone synthetase homologues (bulS1, bulS2) and two SARP regulatory genes (bulY, bulZ). Analysis of the autoregulatory element (ARE)-like sequences by electrophoretic mobility shift assays and footprinting using the purified BulR1 and BulR2 recombinant proteins revealed six ARE regulatory sequences distributed along the bul cluster. These sequences showed specific binding of both BulR1 (the gamma-butyrolactone receptor) and BulR2, a possible pseudo γ-butyrolactone receptor. The protected region in all cases covered a 28-nt sequence with a palindromic structure. Optimal docking area analysis of BulR1 proved that this protein can be presented as either monomer or dimer but not oligomers and that it binds to the conserved ARE sequence in both strands. The effect on tacrolimus production was analysed by deletion of the bulR1 gene, which resulted in a strong decrease of tacrolimus production. Meanwhile, the ΔbulR2 mutation did not affect the biosynthesis of this immunosuppressant.