The conformational features of beta-lactoglobulin, refolded by cooling from a thermally perturbed state, has been characterized by intrinsic and extrinsic fluorescence measurements on the protein. It is found that even at 85-90 degrees C, beta-lactoglobulin does not completely lose its folded structure. The unfolding and refolding of beta-lactoglobulin as observed through intrinsic tryptophan fluorescence is nearly reversible because the native beta-lactoglobulin and its refolded form, following heating and cooling, show nearly identical tryptophan fluorescence properties. However, the fluorescence properties of an extrinsic probe 1-anilino 8-naphthalene sulfonic acid (ANS) for the native and refolded forms are quite different from each other. Significant increase in fluorescence intensity and blue shifts in emission maxima of ANS bound to refolded beta-lactoglobulin is observed compared to that of the native form. Our results indicate that beta-lactoglobulin, refolded after heating to above 70 degrees C, has deep hydrophobic pockets which can be accessed by ANS. These pockets are either nonexistent or inaccessible to ANS in native beta-lactoglobulin. The opening of the central cavity collapses at pH close to the isoelectric pH of the protein. This indicates that electrostatic repulsion is necessary to keep this access open.