The effect of riboflavin-sensitized photooxidation on calf lens βH-crystallin has been investigated by using fluorescence and circular dichroism techniques. βH-Crystallin showed a pronounced change in its tertiary structure (conformation) as manifested in the near-u.v. circular dichroism spectra and fluorescence yield of tryptophan residues. The rate of tryptophan photolysis was significantly diminished under anaerobic conditions, but was not affected appreciably when D2O was used in the reaction mixture instead of H2O. Ferricyanide and ferricytochrome c added to the solution prior to irradiation inhibited the rate of photolysis of tryptophan, suggesting the involvement of O2- anion in the photoreactions. Quantitative assays of O2- and H2O2 in the irradiated protein solution strongly suggest that the Type I photosensitization pathway is involved in the RF-sensitized photooxidation of βH crystallin. The effect of photolysis on the cysteine residues of the protein was also studied. The sulfhydryl specific fluorophore N-iodoacetyl-N′-(5-sulfonaphthyl) ethylenediamine (1,5-IAEDANS) was used to study the change in the microenvironment of the cysteine (sulfhydryl) residues of the protein by photolysis. The results indicate that there is a quantitative loss of IAEDANS labeling sites due to photooxidation as well as structural changes of the protein. Fluorescence lifetime measurements indicate that the probe is bound in two environments-the major one (95%) is exposed and the minor one (5%) hydrophobic. A decrease in the lifetimes of the bound label occurs after photooxidation. However, the relative proportion of the hydrophobic IAEDANS-labeling sites increases in the photooxidized βH crystallin, probably due to the formation of supra-aggregated protein by photolysis.