Ozonolysis of guanosine formed the 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) nucleoside along with trace spiroiminodihydantoin (Sp). On the basis of literature precedent, we propose an unconventional ozone mechanism involving incorporation of only one oxygen atom of O3 to form 2Ih with evolution of singlet oxygen responsible for Sp formation. The increased yield of Sp in the buffered 1O2-stabilizing solvent D2O, formation of 2Ih in a short oligodeoxynucleotide, and 18O-isotope labeling provided evidence to support this mechanism. The elusiveness and challenges of working with 2Ih are described in a series of studies on the significant context effects on the half-life of the 2Ih glycosidic bond.