Yeast rad51 mutants are viable, but extremely sensitive to γ-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51− cells accumulated in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51− cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.