Abstract The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase ®) and for the free enzyme were used. For Assemblase ®, it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed. The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase ® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane. The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.