In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30-40 and 40-50 degrees C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0-4.0. Apparent K-m = 12.0 g L-1 haemoglobin (1.56 mM tyrosine) and V-max = 5220 mu mol (mg protein min)(-1) values were obtained for free pepsin, while apparent K-m = 20.0 g L-1 haemoglobin (2.16 mM tyrosine) and V-max = 2780 mu mol (mg protein min)(-1) values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 degrees C for free pepsin and 50 degrees C for immobilized pepsin. (c) 2005 Elsevier Ltd. All rights reserved.