The enzymatic cleavage of the amide bond of L-leucine-4-nitroanilide by the proteolytic enzyme Pronase E releases 4-nitroaniline. The production of this molecule can be observed by the increasing UV absorption of the solution. A decrease in the rate of hydrolysis reaction was obtained in the presence of different humic substances isolated from water, sediment and soil. This result indicates that humic substances influence enzyme activity by interacting with the applied enzyme. A mathematical relation between the concentration of humic substances and their inhibition effect was used to calculate an effective concentration for which 50% of the maximum effect was observed. The obtained concentration was defined as the EC50 value. The EC50 values of the investigated humic substances were in the range 153 mg l-1 to 6360 mg l-1. The amounts of humic substances used in the experiment were higher than in most aquatic environments. However, in bog water or interstitial water of sediments and soils such large amounts of biologically and ecologically relevant natural organic matter can be expected.