C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.