Double-strand break (DSB) repair pathways catalyze the rejoining of broken chromosomes and the integration of transforming DNAs. These processes have been well characterized in bacteria, fungi, and animals. Plants are generally thought primarily to utilize a non-homologous end joining (NHEJ) pathway to repair DSBs and integrate transgenes, as transforming DNAs with large tracts of homology to the chromosome are integrated at random. In order to test the hypothesis that NHEJ is an important pathway for the repair of DSBs in plants, we isolated T-DNA insertion mutations in the Arabidopsis homologs of the Ku80 and DNA ligase IV genes, required for the initiation and completion, respectively, of NHEJ. Both mutants were hypersensitive to the cytostatic effects of gamma radiation, suggesting that NHEJ is indeed a critical pathway for the repair of DSBs. T-DNA insertion rates were also decreased in the mutants, indicating that Ku80 and DNA ligase IV play an important role in either the mechanism or the regulation of T-DNA integration in Arabidopsis.