Evi-1 was originally identified as a common site of viral integration in murine myeloid tumors. Evi-1 encodes a 120-kDa polypeptide containing 10 zinc finger motifs located in two domains 380 amino acids apart and an acidic domain located carboxy terminal to the second set of zinc fingers. These features suggest that Evi-1 is a site-specific DNA-binding protein involved in the regulation of RNA transcription. We have purified Evi-1 protein from E. coli and have employed a gel shift-polymerase chain reaction method using random oligonucleotides to identify a high-affinity binding site for Evi-1. The consensus sequence for this binding site is TGACAAGATAA. Evi-1 protein specifically protects this motif from DNase I digestion. By searching the nucleotide sequence data bases, we have found this binding site both in sequences 5' to genes in putative or known regulatory regions and within intron sequences.