The adenovirus 12S and 13S E1A proteins have been shown to relieve repression mediated by the cellular transcription factor YY1. The 13S E1A protein not only relieves repression but also activates transcription through YY1 binding sites. In this study, using a variety of in vivo and in vitro assays, we demonstrate that both E1A proteins can bind to YY1, although the 13S E1A protein binds more efficiently than the 12S E1A protein. Two domains on the E1A proteins interact with YY1: an amino-terminal sequence (residues 15 to 35) that is present in both E1A proteins and a domain that includes at least a portion of conserved region 3 (residues 140 to 188) that is present in the 13S but not the 12S E1A protein. Two domains on YY1 interact with E1A proteins: one is contained within residues 54 to 260, and the other is contained within the carboxy-terminal domain of YY1 (residues 332 to 414). Cotransfection of a plasmid expressing carboxy-terminal amino acids 332 to 414 of YY1 fused to the GAL4 DNA-binding domain can inhibit expression from a reporter construct with GAL4 DNA binding sites in its promoter, and inclusion of a third plasmid expressing E1A proteins can relieve the repression. Thus, we find a correlation between the ability of E1A to interact with the carboxy-terminal domain of YY1 and its ability to relieve repression caused by the carboxy-terminal domain of YY1. We propose that E1A proteins normally relieve YY1-mediated transcriptional repression by binding directly to the cellular transcription factor.