The prevailing hypothesis on the mechanism of radiation-induced cell killing identifies the genetic material deoxyribonucleic acid (DNA) as the most important subcellular target at biologically relevant doses. In this review we present new data and summarize the role of the DNA double-strand breaks (dsb) induced by ionizing radiation and DNA dsb rejoining as determinants of cellular radiosensitivity. When cells were irradiated at high dose-rate, two molecular end-points were identified which often correlated with radiosensitivity: (1) the apparent number of DNA dsb induced per Gy per DNA unit and (2) the half-time of the fast component of the DNA dsb rejoining kinetics. These two molecular determinants, not mutually exclusive, may be linked through a common factor such as the conformation of DNA.