An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with D-cellobiose or D-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of H-1-NMR spectra in D2O, the product of D-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included D-cellobiose, L-arabinose, D-xylose and sucrose, but the specificity parameter (k(cat)/K-m) was highest for D-glucose. Two electron accepters were identified, namely 2,6-dichloroindophenol and p-benzoquinone. but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of D-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus.