AvrBs3, the founding member of theXanthomonastranscription-activator-like effectors (TALEs), is translocated into the plant cell where it localizes to the nucleus and acts as transcription factor. The DNA-binding domain of AvrBs3 consists of 17.5 nearly-identical 34 amino acid-repeats. Each repeat specifies binding to one base in the target DNA via amino acid residues 12 and 13 termed repeat variable diresidue (RVD). Natural target sequences of TALEs are generally preceded by a thymine (T0), which is coordinated by a tryptophan residue (W232) in a degenerated repeat upstream of the canonical repeats. To investigate the necessity of T0 and the conserved tryptophan for AvrBs3-mediated gene activation we tested TALE mutant derivatives on target sequences preceded by all possible four bases. In addition, we performed domain swaps with TalC from a rice pathogenicXanthomonasbecause TalC lacks the tryptophan residue, and the TalC target sequence is preceded by cytosine. We show that T0 works best and that T0 specificity depends on the repeat number and overall RVD-composition. T0 and W232 appear to be particularly important if the RVD of the first repeat is HD (‘rep1 effect’). Our findings provide novel insights into the mechanism of T0 recognition by TALE proteins and are important for TALE-based biotechnological applications.