Technical Abstract: The association between biofilm formation and the interactions of Salmonella enterica serovars with cut-Romaine-lettuce leaves was investigated. Biofilm formation by 8 S. enterica serovars was tested on polystyrene microtiter plates in the presence of different growth media. Maximal biofilm mass was commonly developed in Luria-Bertani broth without sodium chloride (LBNS), with five out of eight serovars categorized as strong biofilm producers and three weak biofilm producers. Addition of glucose inhibited biofilm formation by the strong producers, while enhancing its development in two other strains. Attachment to intact leaf tissue revealed that strong biofilm producers attached better than weak producers. Further incubation for 3 days, at 30oC, showed no significant change in the number of viable salmonellae cells. Confocal microscopy and viable count studies revealed that attachment for 2h at 25oC, but not for 18 h at 4oC, displayed preferred attraction to cut-regions of the leaf. Storage of intact lettuce pieces, contaminated with salmonellae for 9 days at 4oC demonstrated only small population change (<1 log). Exposure of Salmonella-contaminated lettuce pieces to acid (pH-3.0) resulted in a mild reduction in the population size, in most serovars, compared to a high susceptibility of the planktonic cells. Our results suggest that biofilm formation on polystyrene in LBNS is related to the capacity of Salmonella serovars to attach to cut-Romaine lettuce leaves. Association of Salmonella with Romaine lettuce leaves facilitates survival of the pathogen and enhances its acid tolerance.