The DNA glycosylase pathway, which requires the sequential action of two enzymes for the incision of DNA, presents a serious problem for the efficient repair of oxidative DNA damage, because it generates genotoxic intermediates such as abasic sites and/or blocking 3'-end groups that must be eliminated by additional steps before DNA repair synthesis can be initiated. Besides the logistical problems, biological evidence hints at the existence of an alternative repair pathway. Mutants of Escherichia coli and mice (ref. 4 and M. Takao et al., personal communication) that are deficient in DNA glycosylases that remove oxidized bases are not sensitive to reactive oxygen species, and the E. coli triple mutant nei, nth, fpg is more radioresistant than the wild-type strain. Here we show that Nfo-like endonucleases nick DNA on the 5' side of various oxidatively damaged bases, generating 3'-hydroxyl and 5'-phosphate termini. Nfo-like endonucleases function next to each of the modified bases that we tested, including 5,6-dihydrothymine, 5,6-dihydrouracil, 5-hydroxyuracil and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine residues. The 3'-hydroxyl terminus provides the proper end for DNA repair synthesis; the dangling damaged nucleotide on the 5' side is then a good substrate for human flap-structure endonuclease and for DNA polymerase I of E. coli.