The hydrophobic fluorescent probe 6-(p-toluidinyl)naphthalene-2-sulfonate, when bound to α-crystallin, shows significantly higher emission than that of the dye in water or bound to the other two crystallins, β and γ. The dissociation constant for the binding is 30.2 × 10−6 M at pH 7. The fluorescence intensity of α-crystallin-bound probe decreases considerably in the presence of guanidine hydrochloride, which is known to disaggregate the subunits of the crystallin. Results indicate that α-crystallin has a large number of hydrophobic sites and that these nonpolar regions are formed upon the association of the subunits to the protein. On the basis of the reactivity of 5,5′-dithiobis(2-nitrobenzoic acid) and the fluorescent sulfhydryl probe 6-(4′-maleimidylanilino)-naphthalene-2-sulfonic acid, it appears that α- and γ-crystallins have three different classes of sulfhydryl groups, two of them accessible and the other inaccessible to the reagents employed. All sulfhydryl groups of β-crystallin, on the other hand, are readily accessible to these reagents and can be represented by one class only. The total sulfhydryl groups in the crystallins were estimated in guanidine hydrochloride by using 5,5′-dithiobis(2-nitrobenzoic acid). Accessible classes of sulfhydryl groups of α-crystallin are in a more nonpolar region than in the other two crystallins, indicated by the blue shift of the fluorescence maxima of the probe (415 and 420 nm of α-crystallin compared with 433 nm of β- and γ-crystallins). This is consistent with the results of steady-state quenching by acrylamide and lifetime measurements. Lifetime quenching studies of N-(iodoacetyl)-N′-(5-sulfo-1-naphthyl)ethylenediamine-labeled β-crystallin reveal that only the major component, 10.2 ns, is quenched by acrylamide, with a quenching constant (kq) of 7.7 × 108 M−1 s−1, while the minor one, 19.6 ns, is inaccessible to the quencher.