The magnitude of diffusional restrictions in reactions of peptide hydrolysis and synthesis was studied with α-chymotrypsin immobilized in two different size commercial glyoxal-agarose gel particles at enzyme loads of 0.25, 0.5 and 1 mg protein/g gel. Such magnitude was evaluated by determining the effectiveness factor. Results showed that the effect of diffusional restrictions was stronger for the reaction of hydrolysis than synthesis, being the effectiveness factor in some cases three times higher. Diffusional restrictions were stronger for the catalysts of larger size and with higher enzyme loads, a more than three-fold decrease in the effectiveness factor being observed when the catalyst particle radius increased four times and close to a three-fold decrease when the enzyme load was increased four times. Enzyme loads and particle sizes for avoiding diffusional restrictions in each of the reactions were determined from a steady-state mass balance to the catalyst particle.