Leuconostoc mesenteroides strain NRRL B-1355 produces the soluble exopolysaccharides alternan and dextran in planktonic cultures. Mutants of this strain are available that are deficient in the production of alternan, dextran, or both. Our recent work demonstrated that biofilms from mutant strains contained insoluble polysaccharides. We now find that the insoluble polysaccharides are composed of d-glucose polymers with contiguous sequences of alpha(1 -> 3) and alpha(1 -> 6) linkages. In addition, planktonic cultures of the wild type also produce this insoluble mixture in association with the cell mass. This material is similar to the insoluble glucan matrix known as mutan formed by cariogenic strains of streptococci. The production of insoluble mutan-like glucans may be more widespread among Leuconostoc spp. than previously recognized.