The neu gene is amplified and its protein product is overexpressed in certain human tumors. The adenovirus 5 E1a gene product and c-myc repress neu transcription. Moreover, expression of E1a in neu-transformed cells leads to decrease in transformation phenotype and metastatic potential. The simian virus 40 large T antigen (LT) shares structural and functional homology with E1a and c-myc, and all three proteins bind to the retinoblastoma gene product, Rb. We found that LT also represses neu expression at the transcriptional level. However, LT represses neu promoter by a different mechanism compared to E1a and c-myc, because the region of the neu promoter mediating repression by LT (-172 to -79) is downstream from the region responding to E1a and c-myc (-312 to -172). In addition, a LT mutant (K1) unable to complex Rb still represses neu promoter activity, indicating that the Rb binding domain of LT is not required for repression of neu. Since K1, unlike LT, does not transform Rat-1 cells but, like LT, represses the neu promoter, we tested whether K1 functions as a transformation suppressor of activated neu oncogene. Focus-forming assays showed that K1 indeed suppresses the strong cell-transforming activity of activated neu.