Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, (S)-HPMPC] is an antiviral drug that has been approved for the treatment of cytomegalovirus retinitis in AIDS patients. Cidofovir also possesses potent activity against human papillomavirus-induced tumors in animal models and patients. We have recently shown that cidofovir inhibits the development of vascular tumors induced by basic fibroblast growth factor (FGF2)-overexpressing endothelial cells (FGF2-T-MAE) in mice. Here, we demonstrate that the inhibitory activity of cidofovir in FGF2-T-MAE cells may result from the specific induction of apoptosis. Cell cycle analysis revealed that cidofovir induces accumulation of cells in the S phase and, upon prolonged treatment, a significant increase in sub-G1 cells, exhibiting a sub-diploid DNA content. Moreover, annexin V binding, an early event in apoptosis induction, was increased in cidofovir-treated FGF2-T-MAE cells. Cidofovir also caused nuclear fragmentation and the activation of caspase-3-like proteases, as evidenced by the cleavage of poly(ADP-ribose)polymerase. In addition, cidofovir treatment of FGF2-T-MAE cells resulted in a pronounced up-regulation of the tumor suppressor protein p53. However, the expression of Bax and Bcl-2 remained unchanged and cidofovir did not induce the release of cytochrome c from the mitochondria. Also, cidofovir did not suppress the phosphorylation of protein kinase B/Akt, a transmitter of anti-apoptotic survival signals, or its downstream regulator Bad, indicating that the Akt pathway is not affected by cidofovir in FGF2-T-MAE cells. However, the compound inhibited the expression of FGF2 and FGF2 signaling through Erk42/44, as shown by Western blot analysis. Our results indicate that cidofovir inhibits the growth of FGF2-T-MAE cells via inhibition of FGF2 expression and signaling, and via the induction of apoptosis. These findings suggest that the clinical use of cidofovir might be expanded to tumors that are not induced by oncogenic viruses.