Fluorescent nucleic acid analogues are valuable tools for studying DNA dynamics, protein-DNA interactions and nucleotide-dependent enzyme activities. The recent development of guanine analogues based on the isoxanthopterin structure offers new opportunities to investigate these dynamics, interactions, and activities. Our combined experimental/theoretical investigation of one such analogue, 6,8-dimethylisoxanthopterin (6,8DMI), has shown that absorbance and fluorescence emission spectra of 6,8DMI shift to lower energies as the pH is increased, yielding pK(a) values of 8.3 and 8.5, respectively. We show that these pH-dependent spectral changes result from protonation/deprotonation of the nitrogen at position 3 of 6,8DMI on the basis of pH-dependent iodide ion quenching of 6,8DMI fluorescence and pH-independent absorption and fluorescence properties of 3,6,8-trimethylisoxanthopterin. Quantum mechanical calculations on the neutral and deprotonated forms of 6,8DMI confirm the observed spectral changes.