Fluorescence lifetime and acrylamide quenching studies on the N'-(iodoacetyl)-N'-(5-sulfo-l-naphthyl)ethylenediamine (l,5-IAEDANS)-labeled sulfhydryl groups of bovine lens α-, βH-, and ϒ-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of the fluorescence-labeled crystallins were 15.2, 14.4, and 13.0 ns, and the quenching rate constant, kq,values were 16.6 X 107, 26.9 X 107, and 32.7 X 107M-1s_1for a-, βH-, and γ-crystallins, respectively. The results indicate that as the polarity of the sulfhydryl site increased (i.e., its lifetime decreased), its accessibility to collisional quenching by acrylamide also increased. The minor decay component of the fluorescence label was not significantly quenched by acrylamide for all three classes of crystallins. When the proteins were irradiated in the presence of methylene blue, in a system generating singlet oxygen, the kqvalue for acrylamide quenching of the major decay component of α-crystallin decreased to zero, while its lifetime decreased to 6 ns. Neither the lifetime nor the kqof α-crystallin recovered completely in the presence of the singlet oxygen quencher sodium azide. Light-induced binding of the photosensitizer methylene blue to the crystallins was observed by absorption spectroscopy. The bound photosensitizer partially quenches the fluorescence lifetime of the N-acetyl-N′-(5-sulfo-l-naphthyl)ethylenediamine (AEDANS) label in irradiated α-crystallin. Further decrease in the lifetime occurs as a result of the singlet oxygen mediated conformational change. The results suggest that the fluorescence lifetime of the AEDANS is fully quenched in the irradiated α-crystallin and there is no further quenching by acrylamide. An increase in the fraction of the minor component of βH-crystallin which was inaccessible to acrylamide quenching was observed after irradiation. There was no effect of irradiation on the kqfor acrylamide quenching of the major component of the decay of AEDANS bound to βH- or 7-crystallins. Static quenching was found to contribute significantly to the steady-state quenching plots of the polar sulfhydryl sites of irradiated α-crystallin and of untreated and irradiated βH-and ϒ-crystallins, but it had no detectable role in the case of untreated α-crystallin. Fluorescence anisotropy of the AEDANS label bound to the crystallins was higher in the irradiated crystallins as compared with the controls.