Technical Abstract: Biofilm formation by Listeria monocytogenes is generally associated with its persistence in the food processing environment. Serotype 1/2a strains make up more than 50% of the total isolates recovered from food and environment, while serotype 4b strains are most often associated with major outbreaks of human listeriosis. Using a microplate assay with crystal violet staining, we examined biofilm formation by 18 strains of each serotype in tryptic soy broth with varying concentrations of glucose (from 0.25% to 10.0%, w/v), sodium chloride (from 0.5% to 7.0%, w/v) and ethanol (from 1% to 5.0%, v/v), and at different temperatures (22.5 °C, 30 °C, and 37 °C). A synergistic effect on biofilm formation was observed for glucose, salt and temperature. The serotype 1/2a strains generally formed higher density biofilms than the 4b strains under most conditions tested. Interestingly, most serotype 4b strains had a higher growth rate that the 1/2a strains, suggesting that growth rate may not be directly related to the capacity in biofilm formation. Crystal violet was found to stain both bacterial cells and biofilm matrix material. The enhancement in biofilm formation by environmental factors was apparently due to the production of extracellular polymeric substances instead of accumulating viable biofilm cells.