Significant influences of sulfur and aza substitution on excited-state dynamics in thymine analogues, 6-aza-2-thiothymine (ATT) and 2-thiothymine (2TT), were intensively studied by means of nanosecond transient absorption and time-resolved luminescence spectroscopy. Transient absorption spectral measurements gave distinct spectral features and sufficiently longer lifetimes for both molecules attributable to the T-1 (pi pi*) state under Ar-saturated condition. Additionally, another long-lived subsequent transient was also observed for ATT, which suggests hydrogen abstraction from a ground-state molecule itself takes place only for triplet ATT. Quantum yields for intersystem crossing (Phi(ISC)) were determined to be unity as well as other pyrimidine analogues we reported previously, and efficient photosensitized singlet oxygen O-2* ((1)Delta(g)) formation was also observed in the presence of dissolved molecular oxygen with a quantum yield (Phi(A)) of 0.69 +/- 0.02 for ATT whereas it almost halved for 2TT. These findings have shown the combination of subtle substitutions can possibly control photophysical or photochemical properties such as O-2* ((1)Delta(g)) formation or reactivity in excited states in addition to the substantial intersystem crossing caused by replacing oxygen O2 in thymine by sulfur (referred to S2 substitution henceforward). The experimental results were corroborated by quantum chemical calculation at the B3LYP/6-31+G(d,p)/PCM level.