Photophysical characteristics of N-substituted C5–C5′-linked dihydrothymine dimers (1a,b[meso], meso compounds of [5R,5′S]-bi-5,6-dihydrothymines; 1a,b[rac], racemic compounds of [5R,5′R]-bi-5,6-dihydrothymines and [5S,5′S]-bi-5,6-dihydrothymines) in aqueous solution with varying contents of less-polar aprotic solvent such as tetrahydrofuran or dioxane have been investigated by UV-absorption, and steady-state and time-resolved fluorescence spectroscopies. Among the C5–C5′-linked dimers, (5R,5′S)-bi-5,6-dihydro-1-methylthymine (1a[meso]) showed a redshifted weak UV-absorption band at 270–350 nm and excimer fluorescence emission at λ_max = 370 nm with a quantum yield (Φ_F) of ∼0.1 in phosphate buffer (pH < 10) at 293 K. Racemic compound of 5,6-dihydro-1-methylthymine dimer (1a[rac]), meso and racemic compounds of 5,6-dihydro-1,3-dimethylthymine dimers (1b[meso] and 1b[rac]) in phosphate buffer were nonfluorescent under similar conditions. The UV-absorption and fluorescence spectral characteristics of 1a[meso] in aqueous solution were interpreted in terms of intramolecular stacking interactions between the dihydropyrimidine chromophores leading to a preferential “closed-shell” conformation both in the ground state and the excited singlet state. In basic solutions at pH > pK_a = 11.7, the fluorescence quantum yield of 1a[meso] decreased due to a dominant “open-shell” conformation resulting from the electrostatic repulsion between the deprotonated dihydrothymine chromophores of 1a[meso] in a dianion form.