Unlabelled: Studies examined the effects of meat-contact material types, inoculation substrate, presence of air at the liquid-solid surface interface during incubation, and incubation substrate on the attachment/transfer and subsequent biofilm formation by Escherichia coli O157:H7 on beef carcass fabrication surface materials. Materials studied as 2 × 5 cm coupons included stainless steel, acetal, polypropylene, and high-density polyethylene. A 6-strain rifampicin-resistant E. coli O157:H7 composite was used to inoculate (6 log CFU/mL, g, or cm²) tryptic soy broth (TSB), beef fat/lean tissue homogenate (FLH), conveyor belt-runoff fluids, ground beef, or beef fat. Coupons of each material were submerged (4 °C, 30 min) in the inoculated fluids or ground beef, or placed between 2 pieces of inoculated beef fat with pressure (20 kg) applied. Attachment/transfer of the pathogen was surface material and substrate dependent, although beef fat appeared to negate differences among surface materials. Beef fat was the most effective (P < 0.05) inoculation substrate, followed by ground beef, FLH, and TSB. Incubation (15 °C, 16 d) of beef fat-inoculated coupons in a beef fat homogenate (pH 4.21) allowed the pathogen to survive and grow on coupon surfaces, with maximal biofilm formation observed between 2 and 8 d of storage and when air was present at the liquid-solid interface. The results indicated that the process of fabricating beef carcasses may be conducive to the attachment of E. coli O157:H7 onto meat-contact surfaces and subsequent biofilm formation. Furthermore, it is recommended that substrates found in beef fabrication settings, rather than laboratory culture media, be used in studies designed to investigate E. coli O157:H7 biofilm development and control in these environments., Practical Application: Findings of this study provide knowledge on the effect of type of beef carcass fabrication surface material, fabrication-floor fluids and residues, and incubation conditions on attachment/transfer and subsequent biofilm formation by E. coli O157:H7. The results highlight the importance of thoroughly cleaning soiled surfaces to remove all remnants of beef fat or other organic material that may harbor or protect microbial contaminants during otherwise lethal antimicrobial interventions., (© 2012 Institute of Food Technologists®)