Lactobacillus reuteri is known to produce exopolysaccharides (EPS), which have the potential to be used as an alternative biothickener in the food industry. In this study, the effect of several environmental conditions on the growth and EPS production in the L. reuteri strain ATCC 55730 was determined. The expression of the corresponding reuteransucrase gene, gtfO, was investigated over time and the results indicated that the expression increased with growth during the exponential phase and subsequently decreased in the stationary phase. Fermentation with glucose and/or sucrose as carbon and energy source revealed that gtfO was constitutively expressed and that the activity profile was independent of the sugar source. In the applied ranges of parameter values, temperature and pH were the most important factors for EPS formation and only temperature for growth. The best EPS yield, 1.4 g g(-1) CDW, was obtained at the conditions 37 degrees C, pH 4.5 and 100 g l(-1) sucrose, which were close to the estimated optimal conditions: pH 4.56 and 100 g l(-1) sucrose. No EPS formation could be detected with glucose. In addition, no direct connection between the expression and the activity of reuteransucrase could be established. Finally, the strain ATCC 55730 was benchmarked against 14 other L. reuteri strains with respect to EPS production from sucrose and abilities to metabolise sucrose, glucose and fructose. Eight strains were able to produce glucan and a corresponding glucansucrase gene was confirmed for each of them.