Different immobilized preparations of yeast invertase were studied for the purpose of refining the mechanism of inactivation of free enzyme. Four immobilization techniques were applied : biospecific adsorption on the concanavalin A-bead cellulose matrix; covalent immobilization on the activated bead cellulose matrix; and cross-linking of the aforementioned preparations with glutaraldehyde. All preparations were assessed with respect to the activity loss during immobilization, thermal stability, and inactivation kinetics. For each preparation, a corresponding inactivation mechanism was verified using the multitemperature data evaluation. The preparations, whose biological activities were strongly modified during immobilization, were shown to be unsuitable for elucidating the inactivation mechanism of free invertase. The biospecifically adsorbed invertase on the concanavalin A-bead cellulose matrix met all criteria for a preparation that should be a convenient model of free enzyme. This preparation was found to inactivate through a three-step series mechanism with the activation energies of individual reactions at 304, 833, and 675 kJ mol(-1). The results obtained in this study complemented an existing mechanism of the inactivation of free invertase.