Chitobiase (EC 3.2.1.29), from the culture filtrate of Trichoderma harzianum, was purified in sequential steps by ammonium sulfate precipitation. Ion exchange chromatography, and gel filtration. The physical and biochemical properties of the enzyme have been determined. The native enzyme has a molecular weight of 118 kDa when determined by gel filtration, and 64 kDa by SDS-PAGE. The enzyme catalyzed the hydrolysis of N,N-diacetylchitobiose and p-nitrophenyl-beta-N-acetyl glucosamine with apparent Km of 575-mu-M and 235-mu-M, respectively. The pH optimum for the enzyme was pH 5.5, and maximum activity was obtained at 50-degrees-C. Glucosamine and N-acetylglyucosamine strongly inhibited the enzyme.