At least two mechanisms of DNA double-strand break (DSB) repair operate in mammalian cells. Homologous recombination, which plays a major role in lower organisms, plays a less significant role in higher organisms. In contrast, the majority of DSBs in mammalian cells are rejoined by a mechanism, termed non-homologous end joining (NHEJ), that does not depend upon extensive regions of homology. This process is also used to rejoin site-specific DSBs introduced during V(D)J recombination. From the analysis of defective rodent mutants, four proteins (Ku70, Ku80, DNA-PKcs and Xrcc4) that function in this process in mammalian cells have been identified. DNA ligase IV is also strongly implicated since it associates strongly with XRCC4, and since DNA ligase IV-deficient yeast are defective in their ability to carry out NHEJ. In S. cerevisiae, Sir2p, Sir3p and Sir4p, three proteins required for transcriptional silencing, are also required for NHEJ. Additionally, the yeast mutants, xrs2, rad50 and mre11, which are defective in meiotic recombination, are also defective in NHEJ. Here I review the evidence implicating these proteins as functioning in NHEJ and discuss their properties and role in other pathways. The significance of DSB repair to clinical radiosensitivity and human disorders is also evaluated.