Six major pathways for DNA repair have been identified. These include (1) DNA repair by direct reversal, (2) base excision repair, (3) mismatch repair, (4) nucleotide excision repair, (5) homologous recombination, and (6) non-homologous end-joining. In addition, several other cellular processes influence the response to DNA damage. The generation of gene-targeted organisms is crucial for assessing the relative contribution of single DNA repair proteins and DNA repair pathways in maintaining genome stability. In particular, the accumulation of DNA damage, mutations and cancer in unexposed gene-targeted animals illuminates the spontaneous load of a particular lesion and the relative significance of a single gene in a specific pathway. Strategies for the generation of gene-targeted mice have been available for 15 years and more than 100 different genes relevant to DNA repair have been targeted. This review describes some important progress made toward understanding spontaneous DNA damage and its repair, exemplified through one, or a few, gene-targeted mice from each major DNA repair pathway.