Cross-linked enzyme crystals (CLECs) of thermolysin exhibit functional characteristics that are superior to those found in soluble or conventionally immobilized enzymes. Thermolysin-CLECs (T-CLECs) are more stable than the native enzyme in water-immiscible organic solvents and in mixtures of water-miscible organic solvents (DMF, THF, acetone) with water. The operational stability of T-CLECs in these solvents has been demonstrated by the repetitive batch synthesis in ethyl acetate of Z-L-Asp-L-PheOMe, the chiral precursor of the artificial sweetener aspartame. We have also found that T-CLECs are stable in ethanol saturated with salts such as LiCl or CaCl2 which are useful solubilizing agents for the separation and purification of insoluble peptides. Peptides of increasing size have been synthesized with the T-CLECs, including coupling PheNH(2) to the oxidized B-chain of insulin, a 30 amino acid peptide. The initial rates of synthetic reactions catalyzed by T-CLECs (V-CLEC) compared with those catalyzed by native enzme (V-sol) are similar up to a heptapeptide. These data suggest that enzymatic peptide coupling using CLECs might present a feasible alternative to traditional methods both in the laboratory and in large scale applications.