The starch-binding domain (SBD) of glucoamylase from Aspergillus niger consists of 110 amino acid residues. There is one disulfide bond, between Cys3 and Cys98. This article summarizes the thermodynamic and kinetic effects of the disulfide bond on the stability and structure of SBD as determined by calorimetric and spectroscopic studies of wild-type SBD and two disulfide-deficient mutants. Deletion of the disulfide bond did not change the native-state structure or the binding ability of SBD with β-cyclodextrin, a substrate analog. Rather, the disulfide bond stabilized the protein mainly by reducing the entropy of the unfolded state. In addition, the disulfide bond prevented SBD from forming a native-like and kinetically-trapped intermediate state. This possibly misfolded intermediate state was formed when the disulfide-deficient mutants were rapidly cooled from a high temperature, and had a long half-life of 11 h at 5 ^oC. The binding parameters of the native and intermediate states with β-cyclodextrin were essentially the same.