We recently described a yeast assay suitable for genetic screening in which simple religation nonhomologous end-joining (NHEJ) and single-strand annealing (SSA) compete for repair of an I-SceI-created double-strand break. Here, the required allele has been introduced into an array of 4781 MATa deletion mutants and each strain screened individually. Two mutants (rad52 and srs2) showed a clear increase in the NHEJ/SSA ratio due to preferential impairment of SSA, but no mutant increased the absolute frequency of NHEJ significantly above the wild-type level. Seven mutants showed a decreased NHEJ/SSA ratio due to frank loss of NHEJ, which corresponded to all known structural/catalytic NHEJ components (yku70, yku80, dnl4, lif1, rad50, mre11, and xrs2); no new mutants in this category were identified. A clearly separable and surprisingly large set of 16 other mutants showed partial defects in NHEJ. Further examination of these revealed that NEJ1 can entirely account for the mating-type regulation of NHEJ, but that this regulatory role was distinct from the postdiauxic/stationary-phase induction of NHEJ that was deficient in other mutants (especially doa1, fyv6, and mck1). These results are discussed in the context of the minimal set of required proteins and regulatory inputs for NHEJ.