In order to combine the high potential of frozen state peptide synthesis and the advantages of the application of immobilized proteases, the capability of carrier-bound cc-chymotrypsin (CT, EC 3.4.21.1) to form peptide bonds in frozen aqueous reaction mixtures was investigated. The properties of the support materials strongly influenced the peptide yields. CT bound to rather hydrophobic supports catalysed peptide synthesis as effectively as the soluble enzyme. The immobilized enzyme preparations could be successfully re-used at least 15 times. Peptide synthesis catalysed by immobilized chymotrypsin was also studied in low water organic reaction mixtures at sub-zero temperatures. The advantages and limitations of the different approaches are discussed.