In order to clarify the mechanism for the peptide synthesis with an immobilized enzyme in a water-immiscible organic solvent system, we studied the synthesis of Z-AspPheOMe from Z-Asp and PheOMe catalyzed by thermolysin immobilized onto Amberlite XAD-7. As an organic solvent, ethyl acetate was used. The reaction was also done in the aqueous/organic biphasic system and in ethyl acetate containing a small amount of water using free enzyme for comparison. The substrate concentration dependencies of the initial rate for the synthesis with the immobilized enzyme in ethyl acetate were quite different from those in aqueous buffer with the free enzyme, but similar to those measured in the biphasic system or in ethyl acetate containing a small amount of water. Therefore, it was considered that as a first approximation the reaction in the water-immiscible organic solvent with the immobilized enzyme could be treated by the aqueous/organic biphasic reaction. Based on this consideration, the optimum reaction condition for the reaction with the immobilized enzyme in ethyl acetate for the synthesis of Z-AspPheOMe could be estimated.