Levansucrase (EC 2.4.1.10) was purified to homogeneity from cell-free extracts of Rahnella aquatilis JCM-1683 by streptomycin treatment, ammonium sulfate fractionation, and HPLC with a DEAE-Toyopearl pak 650M, TSKgel G-3000SW, and TSKgel DEAE-5PW. The enzyme had optimum activity around pH and 55—60°C. The molecular weight of the enzyme was 120,000 on gel filtration and it was composed of two identical subunits (64,000). The amount of levan synthesized by the purified enzyme was 2.95 g from 10.0g sucrose. The enzyme had a broad acceptor specificity and gave transfer products. D-Xylose, D-arabinose, L-arabinose, lactose, maltose, maltotriose, cellobiose, and melibiose were effective acceptors in the transfructosylation reaction of the enzyme.