The ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS-1 recognize different DNA sequences with a core element centered over 5′-GGAA-3′ and 5′-GGAA/T-3′, respectively. To understand the molecular basis of this recognition, we carried out site-directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5′-GGAA-3′ and 5′-GGAT-3′ containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein-DNA interaction with the 5′-GGAA-3′ containing oligonucleotide caused by a faster release of the protein from the DNA (koff tenfold higher than the wild-type protein). With the double mutant A231S-N236Y, we obtained an increase in binding affinity and stability toward both 5′-GGAA-3′ and 5′-GGAT-3′ containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5′-GGAA-3′ and 5′-GGAT-3′ oligonucleotides. The free energy of dissociation for the double mutant A231S-N236Y with 5′-GGAA-3′ (ΔΔG((A231S-N236Y) − (N236Y)) = −1.2 kcal mol−1) confirm the stabilizing effect of this mutant in the protein-DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5′-GGAA-3′ and 5′-GGAT-3′ sequences, while A231S mutation modulates the degree of specificity toward 5′-GGAA-3′ and 5′GGAT-3′ sequences. This study explains why wild-type PU.1 does not recognize 5′-GGAT-3′ sequences and in addition broadens our understanding of 5′-GGAA/T-3′ recognition by ETS protein family members.