Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of α-, β_H- and β_L-crystallins of bovine eye lens. The transient absorption spectra, recorded 5 μs after the laser pulse, reveal the presence of the absorption band (625–675 nm) of the RF neutral triplet state (τ = 42 μs) accompanied by the appearance of a long-lived absorption (τ = 320 μs) in the 500–600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RF-sensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated α-, β_H- and β_L-crystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.