Exopolysaccharide (EPS)-producing lactic cultures (LC) have been used to improve body and texture of fermented dairy products for decades. Research at South Dakota State University indicated that reduced fat Cheddar cheese made with EPS-producing cultures had similar textural and melting properties to its full fat counterpart. Furthermore, whey from such cheeses contained EPS that enhanced the functional properties of whey protein concentrate. We hypothesized that the EPS-producing bacteria surviving whey pasteurization would attach to the separation membrane surface and form biofilm. We also hypothesized that the extent of biofilm formation would depend on the type of EPS. To test our hypotheses, we used EPS-producing thermophilic and mesophilic cultures and their EPS-negative mutants to form biofilm on the retentate side of whey reverse osmosis (RO) membranes. To simulate the composition of concentrated whey expected at the membrane surface, we used 10% solution of 35% protein whey protein concentrate as the growth medium. The role of EPS in microbial attachment in the absence of growth (incubation at 4°C) and biofilm formation was investigated. The relationship between biofilm formation and cell surface characteristics was also determined. Results showed that EPS produced by LC may enhance or interfere with bacterial cell attachment and biofilm formation depending on their molecular characteristics. The growth medium did not affect the tendency of the test strains to form biofilm. Bacterial cell surface charge did not seem to impact attachment or biofilm formation on RO membranes. Generally, high cell surface hydrophobicity was associated with greater biofilm formation. In addition to its role in biofilm formation by the producing strains, EPS from LC could also impact biofilm produced by cocultures. A study with slime-producing spore-forming bacteria supported data from the lactic acid bacteria experiments and showed that the hydrophobicity of the extra polymeric substances, whether it is EPS or polyamino acids, plays an important role in biofilm formation on dairy separation membranes.