An extracellular 104 kDa exo-beta-d-glucosaminidase was purified and characterized from the culture supernatant of Aspergillus fumigatus S-26, which showed exceptionally strong chitosanolytic enzyme activity. The purified enzyme showed optimum pH of 3.0-6.0 and optimum temperature of 50-60 degrees C, and was stable between pH 2.0 and 10.0 and under 35 degrees C. The Km, Vmax, and kcat were determined to be 1.0 mg chitosan/ml, 7.8x10(-8) mol/s/mg protein, and 28.3 s-1, respectively. The exo-beta-D-glucosaminidase was severely inactivated by Cu2+ and Hg2+ at 10 mM. 2-Hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, and p-chloromercuribenzoic acid inhibited the enzyme. The enzyme did not degrade chitin, cellulose, and starch. The exo-beta-D-glucosaminidase did not reduce the viscosity of chitosan solutions at early stage of reaction, suggesting the exo-type of cleavage in polymeric chitosan chains. The exo-beta-D-glucosaminidase liberated only GlcN from chitosan, and GlcN plus the one-residue shortened oligomers from (GlcN)2-7. The exo-beta-D-glucosaminidase exhibited transglycosylation activity, resulting in the one-residue elongated oligomers.