Seven thermosensitive glucoamylase mutants generated by random mutagenesis and expressed in Saccharomyces cerevisiae were sequenced and their inactivation kinetics were determined. Wild-type glucoamylase expressed in S. cerevisiae was more glycosylated and more stable than the native Aspergillus niger enzyme. All mutants had lower free energies of inactivation than wild-type glucoamylase. In the Ala39-->Val, Ala302-->Val and Leu410-->Phe mutants, small hydrophobic residues were replaced by larger ones, showing that increases in size and hydrophobicity of residues included in hydrophobic clusters were destabilizing. The Gly396-->Ser and Gly407-->Asp mutants had very flexible residues replaced by more rigid ones, and this probably induced changes in the backbone conformation that destabilized the protein. The Pro128-->Ser mutation changed a rigid residue in an alpha-helix to a more flexible one, and destabilized the protein by increasing the entropy of the unfolded state. The Ala residue in the Ala442-->Thr mutation is in the highly O-glycosylated region surrounded by hydrophilic residues, where it may be a hydrophobic anchor linking the O-glycosylated arm to the catalytic core. It was replaced by a residue that potentially is O-glycosylated. In five of the seven mutations, residues that were part of hydrophobic microdomains were changed, confirming the importance of the latter in protein stability and structure.