Alignment of 967 reference sequences of the typical 2-Cys peroxiredoxin family of proteins revealed that 10 amino acids were conserved, with over 99% identity. To investigate whether the conserved aspartic acid residues and serine residue affect the peroxidase and chaperone activity of the protein, we prepared yeast TSA1 mutant proteins in which aspartic acids at positions 75 and 103 were replaced by valine or asparagine, and serine at position 73 was replaced by alanine. By non-reducing SDS-PAGE, TSA1 and the S73A, D75V and D75N mutants were detected in dimeric form, whereas the D103V and D103N mutants were detected in various forms, ranging from high molecular-weight to monomeric. Compared with wild type TSA1, the D75N mutant exhibited 50% thioredoxin peroxidase activity, and the S73A and D75V mutants showed 25% activity. However, the D103V and D103N mutants showed no peroxidase activity. All proteins, except for the D103V and D103N mutants, exhibited chaperone activity at 43℃. Our results suggest that the two conserved aspartic acid residues and serine residue of TSA1 play important roles in its thioredoxin peroxidase activity, and D103 plays a critical role in its chaperone activity.