The mismatch repair pathway is responsible for maintaining genomic stability by correcting base-base mismatches and insertion/deletion loops that arise mainly via replication errors. Additionally, the mismatch repair pathway performs a central role in the cellular response to both alkylation and reactive oxygen species induced DNA damage. An important step in mismatch processing is the recruitment of hEXO1, a 5′ to 3′ exonuclease, by hMSH2-hMSH6 to remove the nascent DNA strand. However, very little is currently known about the capacity of hEXO1 to exonucleolytically process damaged DNA bases. Therefore, we examined whether hEXO1 can degrade double-stranded DNA substrates containing alkylated or oxidized nucleotides. Our results demonstrated that hEXO1 is capable of degrading duplex DNA containing an O6-methylguanine (O6-meG) adduct paired with either a C or a T. Additionally, the hMSH2-hMSH6 complex stimulated hEXO1 exonuclease activity on the O6-meG/T and O6-meG/C DNA substrates. In contrast, hEXO1 exonuclease activity was significantly blocked by the presence of an 8-oxoguanine adduct in both single and double stranded DNA substrates. Further, hMSH2-hMSH6 was not able to alleviate the nucleolytic block caused by the 8-oxoguanine adduct in heteroduplex DNA. Environ. Mol. Mutagen. 49:388-398, 2008.