AbstractImidazolone (dIz) is an abundant, highly mutagenic, and rather unstable DNA lesion that can cause dG→dC transversion mutations. dIz is generated in DNA by a variety of oxidative processes such as type I photooxidation. Herein we report the synthesis of a carbocyclic nucleoside analogue of dIz and of DNA containing this stabilized lesion analogue. The carbocyclic modification protects this lesion analogue from anomerization. As the repair of the lesion analogue by DNA glycosylases is not possible, this analogue should allow cocrystallization studies together with wild‐type repair enzymes. Characterization of the lesion analogue was performed by using spectroscopic methods and enzymatic digestion experiments of the oligonucleotides.