A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M-r of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (1) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40degreesC. The inulinase exhibited an apparent K-m value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.