The promoter of the Insulin-like growth factor I (IGF-I) gene is activated by the Simian Virus 40 large T antigen (SVLT), and one of the elements responding to SVLT activation has been localized to a short 124 bp immediately upstream of the first initiation of transcription site. This short promoter contains an E2F binding site, that, in gel shifts, binds a protein complex, but only when the promoter activity is reduced or absent. A mutation in the E2F binding site deregulates the activity of the promoter, which becomes active even in those conditions in which the wild type promoter is inactive. By using antibodies in gel retardation analyses, we can show that the different protein complexes include, at least, the following proteins: E2F, cyclin A and p107. We conclude that the short IGF-I promoter is negatively regulated by an E2F binding site that complexes with several proteins. Our data suggest that disaggregation of these complexes by the action of SVLT (or other activators) increases expression from the promoter, thus establishing a link between the regulation of cell proliferation by growth factors and the E2F-associated proteins.