Bacillus polymyxa β-amylase contains three cysteine residues at positions 83, 91, and 323, which can react with sulfhydryl reagents. To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S, C91V, C323S, and C-free. Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis. A disulfide bond between Cys83 and Cys91 was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91V enzymes followed by amino acid sequencing. Therefore, only Cys323 contains a free SH group. Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the kcat/Km value of the enzyme. C323S, containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme. None of the cysteine residues participate directly in the catalytic reaction.