Conditionally replicating adenoviruses (CRADs) are a novel strategy in cancer treatment and clinical trials using CRADs targeted to tumor cells have been reported recently. We hypothesized that it would be possible to construct CRADs targeted to dividing endothelial cells, which are present in the tumor endothelium. We utilized the regulatory elements of Flk-1 and endoglin genes, which have been shown to be highly overexpressed in angiogenic endothelial cells, to construct two CRADs: Ad.Flk-1, which has adenoviral E1A gene under the control of the Flk-1 enhancer/promoter, and Ad.Flk-Endo, which harbors the same Flk-1 enhancer/promoter as Ad.Flk-1, plus it has the adenoviral E1B gene under control of the endoglin promoter. Viral titer measurements by plaque assay showed that in human umbilical vein endothelial cells (HUVECs), both CRADs replicated at levels comparable to that of wild-type adenovirus. In Flk-1 and endoglin negative Hep3B and A549 cells, however, the replication of Ad.Flk-1 and Ad.Flk-Endo was reduced by 30-fold and 600-fold, respectively. Cytotoxicity assays demonstrated that both CRADs killed HUVECs as effectively as wild-type adenovirus and their cytotoxicity in Hep3B and A549 cells was comparable to nonreplicating control adenovirus. Furthermore, there was a striking inhibition (83-91%) of capillary network formation in an in vitro angiogenesis assay when HUVECs were infected with Ad.Flk-1 or Ad.Flk-Endo as compared with the nonreplicating control virus. These results demonstrate that CRADs can be transcriptionally targeted to dividing endothelial cells with high specificity, and that the combined use of Flk-1 and endoglin regulatory elements has a synergistic effect on targeting specificity. This principle may be incorporated into novel therapeutic agents to develop anti-angiogenic treatment for cancer.