ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone alpha-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of alpha-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4'-Dianilino-1,1'-binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on alpha-crystallin, but no appreciable enhancement of the same was observed for the substrate protein gamma-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mgm concentration stabilizes the alpha-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone alpha-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to alpha-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5'-O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of alpha-crystallin in vivo is discussed.