The unfolding and refolding of the tetrameric D-glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated by correlating the biological activity with the protein transitions as monitored by fluorometry, c.d., and by its retention volumes in molecular sieve chromatography. Treatment of the enzyme with SDS (0.1%) resulted in the dissociation of the tetramer (T) into an active dimer (D) with no gross change in the tertiary structure but with change in the secondary structure. On removal of the denaturant, a pars’ of the dimer reassociated to form a small amount of tetramer. Incubation of the enzyme with Gdn. HCl (2M) resulted in the formation of an inactive dimer (D*) that had conformational properties of a molten globule, namely, a native-like secondary structure and disordered tertiary structure. Regaining of activity was observed on lowering of the concentration of the denaturant by dilution. Refolding of the Gdn-HCl-treated enzyme resulted in the restoration of its tertiary structure and activity. The enzyme was completely inactivated by heating at 100 degrees C for 5 min. The heated enzyme is a monomer and exhibits a distinct irreversible change in its structure. Thus, four distinct species have been identified and characterized during denaturation and renaturation of the GXI : (i) native tetramer (T), (ii) active and inactive dimers (D & D*), and (iii) inactive monomer (M). The results suggest that the intact tertiary rather than the secondary structure is essential for GXI activity.