5-Hydroxyuracil (5-OHU) in DNA, arising during endogenous DNA damage and caused by ionizing radiation, is removed by the base excision repair pathway. However, in addition to base lesions, ionizing radiation also generates DNA single-strand breaks (SSBs). When these DNA lesions are located in the proximity of each other, this may result in a profound effect on both repair of the damaged base and the SSB. We therefore examined the repair of DNA substrates containing 5-OHU lesions in the proximity of the 3'-end of a SSB. We found that SSB repair by DNA ligase IIIalpha and DNA polymerase beta is impaired by the presence of the nearby 5-OHU lesion, indicating the requirement for a DNA glycosylase which would be able to remove 5-OHU before SSB repair. Subsequently, we found that although both SMUG1 and NEIL1 are able to excise 5-OHU lesions located in the proximity of the 3'-end of a DNA SSB, NEIL1 is more efficient in the repair of these DNA lesions.