The cytotoxic effects of thiopurine drugs are mostly exerted through the formation of thioguanine nucleotide and its subsequent incorporation into DNA. The 6-thioguanine (6-TG) in DNA can be converted to S6-methylthio-2-aminopurine (2-AP-6-SCH3) and 2-aminopurine-6-sulfonic acid (2-AP-6-SO3H) upon reaction with S-adenosyl-L-methionine and irradiation with UVA light, respectively. Here we prepared oligodeoxynucleotides (ODNs) harboring a 6-TG, 2-AP-6-SCH3 or 2-AP-6-SO3H at a defined site and examined, by using LC-MS/MS, the in vitro replication of these substrates with yeast polymerase eta and Klenow fragment (KF-). Our results revealed that 2-AP-6-SCH3 could be bypassed by KF-, with significant misincorporation of thymine opposite the lesion. The 2-AP-6-SO3H, however, blocked markedly the nucleotide insertion by KF-. Yeast pol eta could bypass all three modified nucleosides; although dCMP was inserted preferentially, we found substantial misincorporation of dTMP and dAMP opposite 2-AP-6-SCH3 and 2-AP-6-SO3H, respectively. Moreover, both KF- and yeast pol eta induced a considerable amount of -2 frameshift products from the replication of 2-AP-6-SCH3- and 2-AP-6-SO3H-bearing substrates. Our results also underscored the importance of measuring the relative ionization efficiencies of replication products in the accurate quantification of these products by LC-MS/MS. Moreover, thermodynamic studies revealed that 2-AP-6-SCH3 and 2-AP-6-SO3H could cause more destabilization to duplex DNA than 6-TG. Taken together, the results from this study shed important new light on the biological implications of the two metabolites of 6-TG.