Amplification/overexpression of the human neu protooncogene has been frequently found in human primary breast and ovarian cancers and is correlated with the number of axillary lymph nodes positive for metastasis in breast cancer patients. Identification of the factors controlling transcription of the neu gene is essential for understanding the mechanisms of neu gene regulation and its role in tumorigenicity. The adenovirus early region 1A (E1A) gene products are pleiotropic transcription regulators of viral and cellular genes and have been identified as a viral suppressor gene for metastasis. Here we demonstrate that transcription of neu can be strongly repressed by the E1A gene products. The 13S and 12S products of E1A gene are effective at repressing neu transcription and the transcriptional repression requires the conserved region 2 of the E1A proteins. The target for E1A repression was localized within a 139-base-pair DNA fragment in the upstream region of the neu promoter. In addition, competition experiments suggest that the sequence TGGAATG, within the 139-base-pair fragment, is an important element for the E1A-induced repression. These results indicate that E1A negatively regulates neu gene expression at the transcriptional level by means of a specific DNA element.