We have studied the importance of the specific DNA sequence of the deo operator site for DeoR repressor binding by introducing symmetrical, single basepair substitutions at all positions in the deo operator and tested the ability of these variants to titrate DeoR in vivo. Our results show that a 16 bp palindromic sequence constitutes the deo operator. Positions outside this palindrome (positions +/- 9, +/- 10) can be changed without any major effect on DeoR binding. Most of the central 6-8 bp of the palindrome (positions +/- 1, +/- 2, +/- 3) can be substituted with other nucleotides with no or only minor effects on DeoR binding, while changes at position +/- 4 and +/- 5 give a more heterogeneous response. Finally, changes at positions +/- 6, +/- 7 and +/- 8 severely disrupt DeoR binding.