Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.