Biofunctional membranes are entities in which biological molecules (or cells) are attached to polymeric supports cast in the form of porous membranes. Such membranes are gaining increased importance in applications of enzymatic catalysis or synthesis (bioreactors), separations (affinity membranes), and chemical analysis (biosensors). However, fundamental studies of the active site of immobilized biomolecules have been rare. In this study, electron paramagnetic resonance (EPR) spin-labeling techniques using a short, active-site specific spin label have been employed to study the properties of a model enzyme, papain, immobilized on a fully-hydrated, modified polysulfone membrane. The EPR properties of the immobilized enzyme and reaction rate results using the amidase activity of papain and N-benzoyl-DL-arginine-4-nitroanilide hydrochloride as substrate are compared with that of the free enzyme in solution. The major findings in this study are : (1) Immobilization does change the active-site conformation of papain. The spin label at the active site of the immobilized papain has slower motion than that of the free papain in solution. (2) There are two major subpopulations of immobilized enzyme on modified er spin-label motion at the active site than subpopulation D, suggesting that the enzyme in subpopulation A may have a more open active-site cleft than that of the subpopulation D. (3) The active-site conformation of subpopulation D of the immobilized papain is insensitive to the pH of the bulk solution, while that of subpopulation A has a similar response to pH changes as that of free papain in solution, suggesting that subpopulation A may be the active form of the immobilized enzyme while subpopulation D is the denatured form.