We have previously identified two positive regulatory elements in the retinoblastoma gene (RB) promoter. One is an ATF site and the other we have called a retinoblastoma binding factor 1 site. In addition, a consensus E2F site is located directly 3' to the ATF site. Here we demonstrate with gel shift assays that E2F and E2F-dependent complexes can bind in vitro to the E2F site of the RB promoter (RB-E2F site). Moreover, we demonstrate that deletion of the RB-E2F site results in stimulation of RB promoter activity in transient transfection assays in several cell lines. Specific point mutations in the E2F site that inhibit the binding of E2F and its complexes also stimulate the RB promoter activity, suggesting that a factor(s) that can bind to the RB-E2F site could be a silencer. RB promoter activity was also stimulated two- to four-fold by mutation of the E2F site in the RB-negative cell lines, J82 and HTB9. Taken together, our results show that the E2F site in the promoter of the RB tumor-suppressor gene acts as a silencer element and that the silencer effect of the E2F site is not necessarily dependent on the presence of the RB protein.