The retinoblastoma tumor suppressor protein and its family members, p107 and p130, are major regulators of the mammalian cell cycle. They exert their growth suppressive effects at least in part by binding the E2F family of transcription factors and inhibiting their transcriptional activity. Agents that disrupt the interaction between Rb family proteins and E2F promote cell proliferation. Here we describe the characterization of a novel interaction between Rb family proteins and a potential tumor suppressor protein, prohibitin. Prohibitin physically interacts with all three Rb family proteins in vitro and in vivo, and was very effective in repressing E2F-mediated transcription. Prohibitin could inhibit the activity of E2Fs 1, 2, 3, 4 and 5, but could not affect the activity of promoters lacking an E2F site. Surprisingly, prohibitin-mediated repression of E2F could not be reversed by adenovirus E1A protein. A prohibitin mutant that could not bind to Rb was impaired in its ability to repress E2F activity and inhibit cell proliferation. We believe that prohibitin is a novel regulator of E2F activity that responds to specific signaling cascades.