Lens alpha-crystallin has been reported to act like a chaperone molecule, with the chaperone-like activity enhanced by partial unfolding. The nature of the partial unfolding, however, is not fully understood. In this project, the unfolding and refolding process of alpha-crystallin was studied with guanidine hydrochloride (GdnHCl). Trp fluorescence (tertiary structure) and far-ultraviolet circular dichroism (UVCD) (secondary structure) demonstrated the presence of an intermediate in the unfolding pathway. ANS (1-anilino-8-naphthalenesulfonate) fluorescence clearly indicated a two-step transition in the unfolding-refolding process and showed that maximum hydrophobicity of the alpha-crystallin occurred at 0.8-1.0 M GdnHCl. This alpha-crystallin intermediate appears to be in a molten globule state; conformational study by near- and far-UVCD measurements indicated that alpha-crystallin intermediate exhibited tertiary structure which was significantly altered from that of the native protein, but had nearly the same secondary structure. Quaternary structure (size of aggregate) of the intermediate also remained unchanged from that of the native protein, as shown by FPLC size exclusion chromatography. The maximal hydrophobicity of the alpha-crystallin intermediate in the unfolding-refolding pathway was accompanied by maximal protection of betaH-crystallin from aggregation. However, an adverse effect of partial unfolding is that the alpha-crystallin intermediate aggregates at high concentrations. Together, these results clearly demonstrated the biological significance of the alpha-crystallin intermediate: it is a more effective chaperone than native alpha-crystallin.