Radical cations of nucleobases are key intermediates causing genome mutation, among which cytosine C center dot+-is of growing importance because the ensuing cytosine oxidation causes GC -> AT transversions in DNA replication. Although the chemistry and biology of steady-state C oxidation products have been characterized, time-resolved study of initial degradation pathways of C center dot+ is still at the preliminary stage. Herein, we choose i-motif, a unique C-quadruplex structure composed of hemiprotonated base pairs C(H)(+):C, to examine C center dot+ degradation in a DNA surrounding without interference of G bases. Comprehensive time-resolved spectroscopy were performed to track C center dot+ dynamics in i-motif and in free base dC. The competing pathways of deprotonation (1.4 X 10(7) s(-1)), tautomerization (8.8 x 10(4) s(-1)), and hydration (5.3 X 10(3) s(-1)) are differentiated, and their rate constants are determined for the first time, underlining the strong reactivity of C center dot+ Distinct pathway is observed in i-motif compared with dC, showing the prominent features of C center dot+ hydration forming C(50H)(center dot) and C(60H)(center dot). By further experiments of pH-dependence, comparison with single strand, and with Ag+ mediated i-motif, the mechanisms of C center dot+ degradation in i-motif are disclosed. The hydrogen-bonding within C(H)(+):C plays a significant role in guiding the reaction flux, by blocking the tautomerization of C(-H)(center dot) and reversing the equilibrium from C(-H)(center dot) to C center dot+. The C radicals in i-motif thus retain more cation character, and are mainly subject to hydration leading to lesion products that can induce disruption of i-motif structure and affect its critical roles in gene-regulation.