DNA single-strand breaks containing 3'-8-oxoguanine (3'-8-oxoG) ends can arise as a consequence of ionizing radiation and as a result of DNA polymerase infidelity by misincorporation of 8-oxodGMP. In this study we examined the mechanism of repair of 3'-8-oxoG within a single-strand break using purified base excision repair enzymes and human whole cell extracts. We find that 3'-8-oxoG inhibits ligation by DNA ligase IIIalpha or DNA ligase I, inhibits extension by DNA polymerase beta and that the lesion is resistant to excision by DNA glycosylases involved in the repair of oxidative lesions in human cells. However, we find that purified human AP-endonuclease 1 (APE1) is able to remove 3'-8-oxoG lesions. By fractionation of human whole cell extracts and immunoprecipitation of fractions containing 3'-8-oxoG excision activity, we further demonstrate that APE1 is the major activity involved in the repair of 3'-8-oxoG lesions in human cells and finally we reconstituted repair of the 3'-8-oxoG-containing oligonucleotide duplex with purified human enzymes including APE1, DNA polymerase beta and DNA ligase IIIalpha.