This study investigated the role of exopolysaccharide (EPS) in cheese moisture retention. Analysis of low-fat Mozzarella cheese made with different combinations of EPS-producing (Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii ssp. bulgaricus MR-lR) and non-EPS-producing (S. thermophilus TA061 and L. helveticus LH100) starters showed significantly higher moisture levels in cheese made with S. thermophilus MR-1C. To determine if the S. thermophilus MR-1C EPS was responsible for increased moisture retention, gene replacement was used to inactivate the epsE gene in this bacterium. Low-fat Mozzarella cheese made with L. helveticus LH100 plus the EPS-negative mutant, S. thermophilus DM1O, had significantly lower moisture content than cheese made with LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-lC EPS indicated that it had a repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1. Interestingly, carbohydrate utilization tests showed that DMlO had acquired the ability to ferment galactose.