Tetracycline repressor (TetR) bears an unstructured loop region between helices α8 and α9, which is moderately permissive to amino acid exchanges and length variations. Recognition sites for the site-specific recombinases Flp (FRT) or Cre (lox) were inserted in-frame into tetR, substituting some of this loop’s codons. A number of the deduced TetR variants displayed efficient regulation in vivo, thus allowing the establishment of a new mode of TetR activation on the genetic level. Chromosomally encoded tetR in Bacillus subtilis was disrupted and inactivated by insertion of a lox66-aphAIII-lox71 kanamycin resistance cassette. Marker excision by Cre recombinase led to the assembly of a novel tetR allele. The encoded regulator, termed TetRlox72/1, is distinguished from wt-TetR by a slightly elongated and altered α8–α9 loop only, harboring an amino acid stretch encoded by lox72. Despite decreased intracellular protein amounts, TetRlox72/1 displayed efficient in vivo activity in B. subtilis and E. coli, indistinguishable from that of wt-TetR. These results underline the sequence flexibility of TetR in the α8-α9 loop and demonstrate the possible use of the regulator as a read-out tool for the activity of site-specific recombinases.