Abstract: Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.