The TetR PQ39 mutant exhibits a new recognition specificity for the tetO-4C operator, but the affinity is not sufficiently high for use in vivo. A stepwise selection of additional mutations by cassette mutagenesis with randomization of residues in the TetR alpha-helix-turn-alpha-helix motif (HTH) yielded mutant TetR EA37PQ39YM42 showing a similar affinity and increased specificity for tetO-4C as wild-type TetR for tetO. A set of mutants obtained by that approach revealed that the fourth residue of the HTH (Leu41), which points towards the core of the DNA binding domain in TetR, alters the recognition of base-pair 4, e.g. the mutant TetR LV41YM42 exhibits a new recognition specificity for tetO-4G. A small residue at the last position in the turn of the HTH increases the affinity and specificity of DNA binding of TetR mutants containing the PQ39 exchange. Thus, cooperation between residues at positions 37, 39, 41 and 42 in the HTH of TetR is necessary to optimize recognition of base-pair 4. We conclude that creating a new DNA recognition specificity in the HTH of TetR with high affinity for the tetO-4C operator variant requires exchanges altering flexibility and/or adjustment of the recognition alpha-helix to the target DNA in addition to the contacting residue.