The ETS1 protein purified from CEM cells was used to select its optimum DNA binding sequence (pu) G/C C/a G G A A G T/c (py). The sequence CCGGAAGT (ETS1-3) was preferred 5:1 over CAGGAAGT (PEA3). Quantitative electrophoretic mobility shift assays (EMSA) indicate that the purified ETS1 protein binds to either ETS1-3 or PEA3 oligonucleotide probes with high affinity, and that the purified ETS1 has different binding capacities for ETS1-3 and PEA3 oligonucleotide probes. The ETS1 protein binds 2-5 times more to the ETS1-3 than the PEA3 probe. Competitive binding experiments show that the ETS1-3 and PEA3 probes effectively compete for the binding of ETS1-3. However, change of the core DNA-binding sequence from GGAA to AGAA eliminates competition. The human ETS1 protein selected the same DNA sequence from a mixture of random oligonucleotides, as did the Drosophila E74A protein (one of the most divergent members of the ETS family), strongly suggesting that all proteins containing the ETS 85 amino acid domain (sequences which define the ETS family) will bind to the same sequence.