The alpha-crystallin domain is a protein module used as a building block in the assembly of the small heat-shock protein oligomeric structure and might play a central role in their chaperone-like function. The stringent conservation of this domain suggests that it forms a common structural core, i.e., a region of invariant secondary and tertiary structure. The objective of this study is to determine the sequence-specific secondary structure and the folding pattern of this domain in alphaA-crystallin. Sequential cysteine mutants were constructed between residues 60 and 120, expressed in E. coli and purified using anion exchange and gel filtration chromatography. The mutants were then reacted with a sulfhydryl specific paramagnetic spin label and analyzed by EPR spectroscopy. The local environment of each spin label was characterized by determining its mobility with respect to the protein matrix and its accessibility to collisions with a paramagnetic reagent exclusi vely soluble in the aqueous phase. This analysis resulted in an assignment of the structural class along the polypeptide chain. Periodic patterns in the structural classes as a function of the residue number were used to identify the location of secondary structures and their topography. The investigated region contains a three-turn alpha-helix connecting the B and C-terminal domains and two beta-strands separated by a highly solvent-exposed loop.