The purpose of this study was to examine whether changes in the repair capacity of blood cells could be used as a valuable biomarker for radiation exposure. To characterize the repair kinetics in nonirradiated and irradiated cells we first performed in vitro split dose experiments. DNA damage and DNA repair capacity were analysed using the comet assay. Our results showed that the first in vitro irradiation affects the repair system of the cells, resulting in a decreased repair capacity after the second irradiation. Furthermore, the second irradiation results in a large amount of DNA damage in the blood cells. To test whether the analysis of the DNA repair capacity after in vitro irradiation is also a valuable method for in vivo studies of donors exposed to radiation, we analysed the repair capacity of blood cells of two exposed groups: patients subjected to a radioiodine therapy and chronically irradiated volunteers from the Chernobyl region. Both groups also showed a significantly impaired repair capacity indicating a stress on the hematopoietic system. In addition, in the group of the Ukrainians DNA damage after in vitro irradiation was significantly higher than in a control group. These results lead to the presumption that the repair capacity and the DNA damage after in vitro irradiation might be a very useful biological marker for radiation exposure in population monitoring.