The enzyme trehalase exists in a wide variety of organisms, including bacteria, yeasts, filamentous fungi, plants, insects and invertebrates. Acid trehalases from some thermophilic fungi, such as Humicola grisea or Scytalidium thermophilum, possess mixed properties as compared with those of the two conventional types of fungal trehalases, classified as acid and neutral according to their pH optimum. Usually, trehalases are strictly specific for trehalose. Preliminary studies with crude extracts demonstrated that the thermophilic filamentous fungus Humicola insolens is an excellent producer of conidial trehalase with maximal activity at pH 7.0. In the present study, we describe the purification and some properties of the conidial trehalase from H. insolens. The fungus was cultivated on solid medium containing 4.0% oatmeal (Quaker) and 1.8% agar, for 10 days at 40° C. Trehalase activity was assayed in the presence of 5mM trehalose, 50mM Hepes (pH 7.0) and 1mM CaCl₂ and the glucose released was quantified by the 3,5 dinitrosalicylic acid method, or occasionally by the glucose oxidase method. The enzyme was purified by DEAE-cellulose and phenyl-Sepharose chromatography. After the phenyl-Sepharose step the specific activity was 14.7 U/mg protein and 8.4-fold purification was achieved, with a yield of 23.3%. Analysis in PAGE and SDS-PAGE showed a single protein band. The profile of pH activity of the purified enzyme showed two peaks, one at pH 6.0 and other at pH 7.0. However, maximal activity was achieved at pH 7.0. The enzyme was stimulated by calcium and manganese and was completely inhibited by EDTA at 1 mM. AMP (10 mM) slightly stimulated the enzyme (21%) while ADP (10 mM) and ATP (10 mM) drastically inhibited the enzyme. When determined at pH 7.0 the optimum temperature was 60° C and the Km and Vmax values were 1.19 mM and 33.21 U/mg protein, respectively. The purified enzyme was strictly specific for trehalose. The enzyme was located at the conidia surface. To our knowledge this is the first report of a neutral conidial trehalase in filamentous fungi.