Upon dimerization by oxidation, Hsp33 functions as a molecular chaperone in prokaryotes. Previously published structures of both the inactive and active species are of doubtful relevance to the solution conformations since the inactive (reduced) crystal structure was dimeric, while the active (oxidized) species was crystallized with a truncation of its regulation domain. The interdomain contact site of the inactive monomer, identified in this work, is consistent with that previously observed in the reduced dimer crystal. In contrast, fluorescence quenching of the active dimer contradicted the results expected from the domain-swapped fold observed in the truncated dimer crystal. The results of this study provide important new information concerning controversial issues in the activation process of Hsp33.