Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG>HD∼NN NI>NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 50-T. Another surprising observation was that base mismatches at the 50 end of the target site had more disruptive effects on affinity than those at the 30 end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto undescribed polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones. © 2013 The Author(s).