E2F1 is an essential transcription factor that regulates cell-cycle progression and apoptosis. Overexpression of E2F1 sensitizes neoplastic cells to apoptosis and leads to tumor growth suppression, making it an interesting target for anticancer therapy. Use of E2F1 as a therapeutic, however, requires a detailed knowledge of the mechanisms by which it controls cellular proliferation and apoptosis, and of other potential E2F1 activities. In this study, a differential proteome analysis was performed to identify proteins associated with E2F1 activity in inducible p53-deficient Saos-2ERE2F1 osteosarcoma cells. 2-DE revealed a distinct protein profile at 32 h after E2F1 activation. Thirty-three proteins were reproducibly identified as either up-regulated or down-regulated. Proteins were identified by MALDI-MS. They included hitherto unknown E2F1 target proteins of cytoskeletal origin, chaperones, enzymes, proteasomal proteins, and several heterogeneous nuclear ribonucleoproteins, suggesting its role in the ER-stress response, protein degradation, and modulation of pre-mRNA splicing. Protein analysis-derived results were verified by Western blot using representative protein candidates. Thirteen identified proteins were the products of genes known to be cancer related. Thus, proteome analysis provides new information about the complexity of E2F1 activities in human cancer cells that may be considered when using E2F1 as a drug.