This report describes the identification and purification of a nuclear protein from rat liver that binds selectively to DNA sequences associated with several animal virus enhancers. The binding activity was tracked by direct DNase I footprinting through four steps of biochemical fractionation. These procedures led to the identification of a polypeptide species exhibiting an apparent molecular weight of 20 kD that accounts for enhancer binding activity. DNase I and dimethyl sulfate footprinting assays were used to examine the manner in which the purified protein binds to enhancer elements associated with SV40, murine sarcoma virus, and polyoma virus. The results of these assays indicate that the initial interaction established between the 20-kD protein and each viral enhancer occurs via a common DNA sequence known as the enhancer core homology.