The use of immobilized enzymes, such as oxidoreductases and hydrolases, in contaminated soils offers a simple, efficient, and inexpensive method for solving pollution problems in the soil environment. Investigations on the behavior of a laccase (from the fungus Trametes versicolor), a peroxidase (from horseradish [Armoracia rusticana P. Gaertner, Meyer & Scherb.]), and an acid phosphatase (from wheat [Triticum aestivum L.] germ) were conducted in the presence of sand, various soils, and soil-sand mixtures. The enzymes were either free or immobilized on three natural supports (a montmorillonite, a kaolinite, and a silt loam soil). On immobilization, laccase and peroxidase retained high activities, whereas immobilized acid phosphatase showed reduced activity. The affinity of laccase and peroxidase for their substrates remained practically unchanged (similar Michaelis constant [Km] values) and higher Km values (low substrate affinity) were obtained for immobilized acid phosphatase. By increasing the amount of soil in the soil-sand mixtures or the content of organic matter in soil, an inhibitory effect on the activity of free and immobilized enzymes was observed. The reusability of immobilized laccase and peroxidase, both in the absence and presence of soil, was evaluated by repeated incubation cycles with 2,4-dichlorophenol. After several incubation cycles, the immobilized enzymes retained different activity levels. Furthermore, in the presence of soils, the inhibitory effect decreased after each incubation cycle, indicating that the observed inhibition was, at least in part, due to soluble soil components easily extracted with the buffered substrate solution. Our results indicate that immobilized enzymes introduced into the soil environment have catalytic activity, but their performance is affected by organic or inorganic soil constituents.