12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces human adenovirus (Ad) early region 1A (E1A) messenger ribonucleic acid expression in infected or Ad-transformed cells. Here, we report that deletion analysis has identified a TPA-responsive element (TRE) in the E1A enhancer region. Deletion analysis indicates that the TRE is located upstream of the E1A cap site between nucleotides −237 and −47. Incubation of extracts from TPA-treated cells with radioactively labeled deoxyribonucleic acid (DNA) fragments containing the TRE (−237 to −47) form specific DNA–protein complexes as demonstrated by gel shift analysis and Southwestern blotting. These experiments provide evidence that novel protein–DNA complexes are formed on a region of the E1A promoter required for TPA-enhanced expression. We speculate that these DNA-binding proteins may interact with the TRE and play a critical role in the mechanism through which TPA upregulates transcription from the Ad E1A gene.