Hydration of interfaces is a major determinant of target specificity in protein/DNA interactions. Interfacial hydration is a highly variable feature in DNA recognition by ETS transcription factors and functionally relates to cellular responses to osmotic stress. To understand how hydration is mediated in the conserved ETS/DNA binding interface， secondary structures comprising the DNA contact surface of the strongly hydrated ETS member PU.1 were substituted， one at a time， with corresponding elements from its sparsely hydrated relative Ets-1. The resultant PU.1/Ets-1 chimeras exhibited variably reduced sensitivity to osmotic pressure， indicative of a distributed pattern of interfacial hydration in wildt-ype PU.1. With the exception of the recognition helix H3， the chimeras retained substantially high affinities. Ets-1 residues could therefore offset the loss of favorable hydration contributions in PU.1 via low-water interactions， but at the cost of decreased selectivity at base positions flanking the 5'-GGA-3' core consensus. Substitutions within H3 alone， which contacts the core consensus， impaired binding affinity and PU.1 transactivation in accordance with the evolutionary separation of the chimeric residues involved. The combined biophysical， bioinformatics and functional data therefore supports hydration as an evolved specificity determinant that endows PU.1 with more stringent sequence selection over its ancestral relative Ets-1.