Four experiments were conducted in commercial beef-packing facilities The objectives of these experiments were to: (i) determine and validate a carcass sampling technique and location to determine if central nervous system (CNS) cross-contamination exists/occurs; (ii) determine if residual CNS tissue contamination remains on splitting saws after sanitation procedures; (iii) determine the prevalence of CNS cross-contamination in commercial slaughter facilities; (iv) determine whether washing treatments reduce or eliminate CNS tissue presence in carcass-splitting saws; (v) determine the effectiveness of commercial spray-washing systems in removing CNS tissue from beef carcasses; and (vi) compare residual CNS tissue levels on the blade and in the housings of the Jarvis Buster IX and Buster IV carcass-splitting saws. CNS tissue remained, albeit at very low levels, in the housings and on the blades of carcass-splitting saws after carcass splitting and operational sanitation. Additionally, after splitting carcasses, CNS tissue remaining in the splitting saw housings and on saw blades was found to cross-contaminate subsequent carcasses during splitting. Most splitting saw operational sanitation procedures reduced the amount of CNS tissue remaining in the splitting saw housings and on splitting saw blades, but no treatment eliminated CNS tissue from either to levels below the detection limit of the assay (6 ng/100 cm2). Washing in carcass spray-washing cabinets at three of the five commercial beef-packing facilities reduced, but did not eliminate, presence of CNS tissue in the aitch bone area of carcasses. Carcass spray washing in cabinets at three of the five facilities reduced (P < 0.05) the concentration of CNS tissue in the fourth thoracic vertebra area. While extremely low concentrations of CNS tissue remained in the splitting saw housings, on the splitting saw blades, and on carcasses, it is unknown whether these levels would pose a human food safety risk because the exact amount of bovine spongiform encephalopathy-infected spinal cord capable of transmitting the disease to humans is dependent on the infectivity titer, which is not readily known.