Exopolysaccharides (EPS) produced in situ by sourdough lactobacilli affect rheological properties of dough as well as bread quality and may serve as prebiotics. The aim of this study was to characterize EPS-formation by Lactobacillus sanfranciscensis TMW 1.392 at the molecular level. A levansucrase gene from L. sanfranciscensis TMW 1.392 encompassing 2,300 bp was sequenced. This levansucrase is predicted to be a cell-wall associated protein of 879 amino acids with a relative molecular weight (M-R) of 90,000. The levansucrase gene was heterologously expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme exhibited transferase and hydrolase activities and produced glucose, fructose, 1-kestose and levan from sucrose; truncation of the N-terminal domain did not affect catalytic activity. Kestose formation was enhanced relative to fructose and levan formation by low temperature or high sucrose levels. During growth in wheat doughs, strain TMW 1.392 utilized sucrose to form fructose, 1-kestose, and fructan, whereas a levansucrase deletion mutant, L. sanfranciscensis TMW 1392Deltalev, lost the ability to hydrolyze sucrose, and did not produce fructan or 1-kestose. These results indicate that, in L. sanfranciscensis TMW 1.392, sucrose metabolism and formation of fructan and 1-kestose is dependent on the activity of a single enzyme, levansucrase.