alpha-Crystallin, composed of the subunits alpha A and alpha B, is a major vertebrate eye lens protein, accomplishing a structural role in maintaining lens stability and transparency. Both subunits also occur in low amounts outside the lens, where their precise function is unknown. They are structurally related to the small heat-shock proteins (HSPs), and increasing evidence indicates that they have also functional similarities with the small HSPs. To extend our insight into these structural and functional relationships, the mouse small HSP (HSP25) was compared with bovine alpha-crystallin, with respect to several known properties of the latter. We show that alpha-crystallin and HSP25 resemble each other in secondary structure and have similar stability toward urea dissociation at pH 7.0. Mixed polymers can be formed from any combination of alpha A-crystallin, alpha B-crystallin, and HSP25 subunits. Furthermore, we demonstrate that HSP25, like alpha-crystallin, can function as a molecular chaperone, by suppressing heat-induced aggregation of other proteins, and is an efficient inhibitor of elastase. Finally, HSP25 is found to be a substrate for protein cross-linking by tissue-type transglutaminase, like alpha B-crystallin. Our results thus corroborate that alpha-crystallin and the small HSPs have comparable functions, probably being involved in the protection of other proteins under conditions of stress.