The study of digestive enzymes, especially in important pests like Chilo suppressalis Walker (Lepidoptera: Pyralidae), which are a key constraint on rice production in a wide area of the globe and also in Iran, could be a successful procedure in the development of a safe and useful control strategy. Glycosidase are a type of digestive enzymes which have a critical role in the final stages of carbohydrate digestion; they hydrolyze ⍺-D-(1,4)-glucose linkage such as p-nitrophenyl-⍺-D-glucoside in di and oligosaccharide components. Laboratory reared 4th instar larvae were randomly selected; midgut and salivary gland were removed by dissection under a stereo microscope and glucosidase activities were assayed by Ferreira and Terra’s procedures. The activities of ⍺- and β-glucosidase in the midgut and salivary gland were 0.009, 0.0063, 0.005 and 0.003 μmol/min/mg protein, respectively. The optimal pH and temperature for enzyme activity were determined to be 9 and 45°C for the glucosidases measured, values which are in agreement with other reports, especially in lepidopteran insects, which give values between 8–12 and 20–50°C. The enzyme activity increased with the addition of NaCl, MgCl₂ and CaCl₂ and decreased due to the use of different concentrations of KCl, Urea, EDTA, SDS and Urea both in midgut and the salivary glands. Control of pests by using resistant varieties is one of the most important practices that are dependent on inhibitors in plants. Hence, characterization of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful, on the one hand for a better understanding of enzyme roles in the nutrition physiology of insects, and on the other hand to reach safe and useful controls of insect pests. To cite this article: A. Zibaee et al., C. R. Biologies 332 (2009).