To quantify the singlet oxygen production, nanosecond near-infrared emission spectroscopy was utilized by monitoring its emission at 1270 nm in deuterium oxide (D 2 O) and ACN solutions. Figures and present the decay traces of singlet oxygen emission for dTAT1 and the phenalenone standard in D 2 O and ACN respectively, while Table summarizes the corresponding quantum yields. In air-saturated and O 2 -saturated D 2 O solutions, relatively low singlet oxygen quantum yields of 0.17 ± 0.03 and 0.31 ± 0.04, respectively, were measured. A significantly higher singlet oxygen quantum yield of 0.48 ± 0.05 was measured for dTAT1 in both air and O 2 -saturated ACN solutions. The lower quantum yield of singlet oxygen in aqueous solutions is predominantly caused by the ~7 times lower O 2 solubility in aqueous solution compared to ACN combined with the relatively short triplet lifetime of dTAA1. Especially in air saturated aqueous solutions ([O 2 ] ~ 0.28 mM) only a fraction of dTAT1 triplet states is quenched by O 2 during their short lifetime. The slightly lower bimolecular rate constant of quenching of dTAT1 triplet states by O 2 in aqueous solutions compared to ACN contributes further to the lower singlet oxygen quantum yields in aqueous solutions. In addition, the low singlet oxygen yield of dTAT1 in PBS can be due in part to the generation of other ROS such as superoxide through a competitive electron transfer process from the T 1 (ππ*) state to O 2 . The generation of superoxide has been reported for other thionated nucleobase derivatives in PBS, and is supported by the cell biology experiments reported next.