A 1-FEH II (1-fructan exohydrolase, EC 188.8.131.52) was purified from forced chicory roots (Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A (Con A) affinity chromatography and anion and cation exchange chromatography. This protocol produced a 70-fold purification and a specific activity of 52 nkat mg−1 protein. The apparent size of the enzyme was 60 kDa as estimated by gel filtration and 64 kDa on SDS-PAGE. Optimal activity was found between pH 5.0 and 5.5. The temperature optimum was around 35°C. No product other than fructose could be detected with inulin as the substrate. The purified enzyme exhibited hyperbolic saturation kinetics with an apparent Km of 58 mM for 1-kestose (Kes) and 64 mM for 1,1-nystose (Nys). The purified 1-FEH II hydrolyzed the β(2?1) linkages in inulin, Kes and Nys at rates at least 5 times faster than the β(2?6) linkages in levan oligosaccharides and levanbiose. Fructose did not affect the 1-FEH II activity but sucrose (Suc) was a strong inhibitor of this 1-FEH II (Ki=5.9 mM). The enzyme was partially inhibited by Na-EDTA and CaCl2 (1 mM).