Abstract The reaction of trypsin with N α-carbobenzoxy-L-lysine p-nitroanilide has been studied in the 0 to −60°C temperature region, over a wide range of pH ★ values, using aqueous-dimethyl sulfoxide cryosolvent. Spectrophotometric and kinetic evidence for the detection and stabilization of a tetrahedral intermediate is presented. The rate of formation of this intermediate, and the concentration accumulated, were pH ★-dependent. At pH ★ ≤ 6 (< −40°C) essentially no tetrahedral adduct is formed or accumulated. Instead another intermediate, resembling the substrate in its spectral properties, is detected.