The application of immobilized alpha-chymotrypsin for the purpose of enantioselective hydrolysis of a Schiff's base of D,L-Phe-OEt (D,L-SBPH) in the mixed water-acetonitrile media with the different content of water is described. The immobilized biocatalyst was prepared by the chemical coupling of the enzyme to poly(vinyl alcohol) (PVA) cryogel-the macroporous hydrogel prepared by means of the freezing-thawing techniques. SBPH is water insoluble, and, therefore, acetonitrile (MeCN) with minor water additives was used as a solvent for the reaction of enantioselective hydrolysis of the racemic substrate. The process was conducted for 96-200 h, and L-Phe with the purity up to 98% e.e. precipitated in both the reaction medium and gel-carrier bulk. The product was recovered by washing the organo-insoluble sediment with aqueous ammonia. D-Phe with the purity up to 85% e.e. was recovered from the organic solution of D-ester after its acidic hydrolysis. The PVA-cryogel-attached enzyme was effective in SBPH hydrolysis in MeCN/water mixtures. The immobilized biocatalyst was active for more than 1 mo of application and could be successfully used after another 4 mo storage at +10 degrees C.