The initial events of angiogenesis comprise endothelial cell activation, migration and proliferation. The characteristics of retinal endothelial cells and capillaries are significantly altered in a number of diseases including cancer. Since radiation has been shown as a useful tool in radiotherapy by altering the proliferative changes, it is important to evaluate the responses of the endothelial cells and the capillary network to radiation. We quantified functional and kinetic responses of endothelial cells and capillaries to radiation in an in vitro model. An in vitro angiogenesis model was introduced in our study with endothelial cells cultured on an extracellular matrix gel in which hollow tube-like structures could be rapidly formed. Vessel formation was quantified using stereological techniques. The cell cycle kinetics of endothelial cells and accumulation of DNA damages after radiation were measured using laser scanning cytometry. To study the response of proliferative capillary-like structures to radiation, the vessel network was irradiated with 2 Gy. To evaluate functional and kinetic responses and differentiation of endothelial cells to radiation, cells were irradiated with 2 and 6 Gy. Progressive time- and dose-dependent loss of endothelial cells occurred starting 24 hours after radiation. Vessel growth was significantly retarded at higher dose. A significant percentage of DNA breaks were detected dose-dependently. A large percentage of G1 cells were measured in the irradiated endothelial cell population when compare to the respective sham-treated control population.. These results indicate radiation-induced endothelial cell injuries destroy the integrity of vascular structure. We postulated that apoptosis may represent a biologically relevant mechanism of radiation-induced endothelial cell damage.