The photoreaction between riboflavin tetraacetate and nucleosides was investigated using time-resolved infrared spectroscopy (TRIR), laser flash photolysis with UV-vis detection, fluorescence quenching, absorption spectroscopy, and density functional theory calculations. Riboflavin tetraacetate (RBTA) was studied experimentally with indole and with Shen and Foote's organic soluble silylated guanosine (G'). Lumiflavin and (R)-2-amino-(S)-4-hydroxy-(R)-5-(hydroxymethyl)-tetrahydrofuran were used as computational models for RBTA and for the sugar moiety of the nucleoside, respectively, using density functional theory calculations (B3LYP/6-31G* and B3LYP/6-31+G**). Vibrational spectra were also calculated for the transient species. Time-resolved infrared spectroscopic data obtained using RBTA are in excellent agreement with the calculated spectra for the triplet flavin, and in the presence of silylated guanosine, with the formation of the most stable hydroflavin radical, RBTH, by an electron transfer-proton transfer mechanism. Although the gas-phase calculations indicate that abstraction of a hydrogen atom from the sugar is slightly exothermic, this reaction does not proceed at a rate measurable as monitored by TRIR spectroscopy. Singlet RBTA is also quenched by G' in methylene chloride. Stern-Volmer analysis of the fluorescence quenching data indicates that this reaction proceeds with a rate constant of 8.7 x 108 M-1 s(-1).