ABSTRACT: This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E. coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria–Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene (Salmonella) coupons. At 0, 14 (Salmonella), 24, 48, and 72 h of storage (25 °C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E. coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S. Typhimurium DT104 on biofilms were higher (P < 0.05) in LB than those in LBG, while the E. coli O157:H7 strain showed no difference (P≥ 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S. Typhimurium DT104 and E. coli O157:H7 strains produced higher (P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S. Thompson and E. coli O157:H7 strains were not different (P≥ 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.