The c-myc promoter was investigated as a possible cellular target for SV40 large T (LT) antigen. In fibroblast and epithelial cell lines, the human c-myc promoter was transactivated by LT. This transactivation was dependent of the interaction of LT with the retinoblastoma (RB) protein. The use of deletions and point mutations of the c-myc promoter demonstrated that in both cell types, the E2F binding sites are necessary for such transactivation. Unexpectedly however, over-expression of RB caused an overall transcriptional activation of the c-myc promoter. We resolved this apparent paradox by demonstrating that this activation is a combination of two antagonistic effects: transcriptional repression mediated by the E2F factor, and transcriptional activation independent of this factor. RB was also found to prevent LT-mediated transactivation, and LT inhibited RB-mediated activation independently of the E2F factor. LT therefore antagonizes both the transcriptional repression and activation mediated by RB.