In a screening of inulinolytic fungi, Penicillium sp. TN-88 was selected as the best producer of inulinase. This strain showed a high inulinase productivity of 9.9 U/ml with a ratio of inulinase activity to invertase activity (I/S ratio) of 11.2 in the culture filtrate when grown in a liquid medium containing inulin as the carbon source at 30 degrees C for 4 d. An endoinulinase, P-II, was 45-fold purified from the culture filtrate by DEAE-Cellulofine A-500 and Q-Sepharose HP chromatographies. The enzyme was homogeneous, as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M-r of 68.0 kDa. The specific activity was 105 U/mg. The enzyme activity was highest at pH 5.2 and 50 degrees C. Endoinulinase P-II hydrolyzed inulin to the extent of 70% and liberated inulotriose as the main product, but lacked activity toward sucrose, raffinose or levan. The enzyme activity was completely inactivated by Ag+, Hg2+ or p-chloromercuribenzoate and by chemical modification with carbodiimide or N-bromosuccinimide. The apparent K-m value for inulin was 0.20 mM at 40 degrees C and pH 5.0. The N-terminal amino acid sequence of the enzyme was 1-DDYRPAFHFC PAENXMNEPN GLIQIXSTXH-30.