p50E4F is a cellular transcription factor whose DNA binding activity is stimulated in a phosphorylation-dependent manner by products of the adenovirus E1A oncogene. Although p50E4F does not contain a bZIP DNA binding motif, it binds a tandemly repeated palindromic sequence in the adenovirus E4 promoter that is recognized by a large number of bZIP proteins, but with much greater stability. Analysis of deletions in the p50E4F sequence identified the regions that are responsible for its unique DNA binding properties. Sequence-specific DNA binding and factor dimerization were localized to a C-terminal region containing two C2H2and one CCHC zinc finger motifs; the phosphorylation site critical for DNA binding activity was also localized to this domain. The high stability of p50E4F binding also required residues within the first 83 amino acids of the N-terminus. Analysis of single and double amino acid substitutions in the C-terminal zinc finger domain demonstrated that while the second C2H2zinc finger was required for DNA binding activity, the putative structures of the first C2H2and the CCHC zinc fingers were not. Instead, residues from these other zinc finger motifs appeared to participate in an alternative structure that mediates DNA binding activity and is regulated by phosphorylation.