A novel cellobiase (Cba2) was purified from the culture supernatant of Cellulomonas biazotea and characterized. Cba2 appeared to be a major secretory cellobiase in C. biazotea as its enzymatic activity was estimated to represent over 40% of the total extracellular beta -glucosidase activity. The enzyme was purified over 260-fold subsequent to ammonium sulfate precipitation, gel-filtration chromatography, anion-exchange chromatography, and reversed-phase high-performance liquid chromatography. Cba2 was shown by SDS-PAGE to have a large molecular mass of 109 kDa, which makes it one of the largest secretory cellobiases characterized. Its homogeneity was confirmed by N-terminal amino acid sequencing. The Km and Vma values were 0.025 mM and 0.0048 mM min(-1), respectively, for the Cba2 hydrolysis of p-nitrophenyl-beta -D-glucopyranoside, and 0.73 mM and 0.00033 mM min(-1), respectively, for the hydrolysis of cellobiose (at 37 degreesC and pH 7.0). The purified enzyme has a pH optimum of 4.8 and the optimum temperature for activity is 70 degreesC. In view of the secretory nature of Cba2 and the fact that it is a major component of secretory cellobiases of C. biazotea, it is potentially important in the enzymatic degradation of cellulose, and its availability as a recombinant protein may facilitate the studies of its biotechnological. applications.