Soybean β-amylase was modified with 2, 3-epoxypropyl α-D-[U-¹⁴C]glucopyranoside ([¹⁴C]α-EPG), a radioactive affinity-labeling reagent for β-amylase, until it lost 95% of its enzyme activity. After S-carboxymethylation at pH 8.0 of SH groups, the modified enzyme was digested at pH 7.0 with Achromobacter protease I and the digest was fractionated by reverse-phase HPLC. A radioactive peptide was finally isolated and its amino acid sequence was determined to be ¹⁸¹Leu-Gly-Pro-Ala-Gly-Glu¹⁸⁶. Radioactivity derived from [¹⁴C]-α-EPG was found exclusively at Glu-186, the γ-carboxyl group of which is esterified with the affinity label. It was concluded that the carboxylate of Glu-186 is a functional group at the catalytic site of soybean β-amylase.