1. The C-8 proton of 7-methylguanosine rapidly exchanges with the solvent. The half-life of deuterium exchange is 5.5 min at pH 4.1, 28°, while at pH 7 the exchange is too fast to be measured by NMR spectroscopy. 1,7-Dimethylguanosine and 7-methylinosine behave analogously. The mechanism of this exchange seems to involve acidic dissociation of the C-8 proton. Two mechanistic alternatives, namely, tautomerism and reversible hydrolytic opening of the imidazole ring can be ruled out. This behaviour of 7-methylguanosine is analogous to that of compounds related to thiamine. 2. 7-Methylation of guanine residues in DNA results in a similar rapid isotope exchange at C-8. This was shown by methylating [8- 3H]guanine-labeled DNA with dimethylsulfate. The amount of tritium released from the DNA as tritiated water corresponded to the amount of 7-methylguanine. 3. This observation provides a simple and selective method for the determination of the extent of 7-methylation of guanine residues in alkylated nucleic acids.