Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca2+ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and Futterer, K. (2003) Nat. Struct. Biol. 10, 935-941] has provided evidence for the presence of a bound metal ion, most likely Ca2+. Characterization of site-directed mutants in the putative Ca2+ ion-binding sites of Lb. reuteri Lev and Inu revealed that the Inu Asp520 and Lev Asp500 residues play an important role in Ca2+ binding. Sequence alignments of family GH68 proteins showed that this Ca2+ ion-binding site is (largely) present only in proteins of Gram-positive origin. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Molecular Sequence Numbers: GENBANK: AF459437, AF465251; Chemicals / CAS: calcium ion, 14127-61-8; fructosyltransferase, 9031-67-8; levansucrase, 9030-17-5; Calcium, 7440-70-2; Edetic Acid, 60-00-4; Hexosyltransferases, EC 2.4.1.-; inulosucrase, EC 2.4.1.9; levansucrase, EC 2.4.1.10