Oxidation of guanine by reactive oxygen species and high valent metals produces damaging DNA base lesions like 8-oxo-7,8-dihydroguanine (8-oxoG). 8-oxoG can be further oxidized to form the spiroiminodihydantoin (Sp) lesion, which is even more mutagenic. DNA polymerases preferentially incorporate purines opposite the Sp lesion, and DNA glycosylases excise the Sp lesion from the duplex, although the rate of repair is different for the two Sp diastereomers. To further understand the biological processing of the Sp lesion, differential scanning calorimetry studies were performed on a series of 15-mer DNA duplexes. The thermal and thermodynamic stabilities of each of the Sp diastereomers paired to the four standard DNA bases were investigated. It was found that, regardless of the base-pairing partner, the Sp lesion was always highly destabilizing in terms of DNA melting temperature, enthalpic stability, and overall duplex free energy. We found no significant differences between the two Sp diastereomers, but changing the base-pairing partner of the Sp lesion produced slight differences in stability. Specifically, duplexes with Sp:C pairings were always the most destabilized, whereas pairing the Sp lesion with a purine base modestly increased stability. Overall, these results suggest that, although the stability of the Sp diastereomers cannot explain the differences in the rates of repair by DNA glycosylases, the most stable base-pairing partners do correspond with the nucleotide preference of DNA polymerases.