We recently demonstrated that Drosophila ribosomal protein S3 specifically cleaved duplex oligodeoxynucleotides at sites of 7,8-dihydro-8-oxoguanine (8-oxoGua), presumably due to S3 protein possessing an N-glycosylase activity that is associated with its known apurinic/apyrimidinic (AP) lyase activity. Here we show, using DNA substrates prepared by gamma-irradiation under N2O and analyzed by gas chromatography/isotope-dilution mass spectrometry, that S3 protein efficiently liberates 8-oxoGua as a free base from the damaged DNA substrate. Of the 15 additional modified bases present in the DNA substrate, the only other one acted on by S3 protein was 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Specificity constants measured for the removal of 8-oxoGua and FapyGua indicate that S3 protein has a similar preference for both of these modified purines. Having established that S3 protein contains an N-glycosylase activity, we next examined the repair of duplex oligonucleotides containing 8-oxoGua (8-oxoGua-37-mer) positioned opposite Cyt, Gua, Thy, or Ade. Significant cleavage of the 8-oxoGua-37-mer was only detected for an opposing Cyt. Moreover, we show that an imino covalent enzyme-substrate intermediate is formed between S3 protein and 8-oxoGua-37-mer, a result similar to other DNA repair enzymes that catalyze N-glycosylase/AP lyase-type reactions at sites of DNA damage.