Aims: The purpose of this study was to develop an effective method for detecting prion (PrP) antigenic determinants remaining in bovine meat and bone meal (MBM) using pressurized fluid extraction (PSE) equipment and flow microbead immunoassay (FMI). Methods and Results: Using the FMI, bovine recombinant PrP could be determined quantitatively in the 7 pmol-7 nmol range using anti-PrP peptide polyclonal antibody-coupled microbeads and anti-PrP monoclonal antibody (SAF61) as a detection antibody. PSE extraction at 120 degrees C for 5 min under high pressure was most effective for eluting PrP determinants from bovine MBMs. The FMI was capable of detecting PrP determinants in bovine MBM extracts with high specificity and indicated that the MBMs contained high levels of PrP determinants. This assay was also applied to the detection of PrPSc determinants in bovine MBM spiked with a scrapie-infected brain at a weight ratio of 50 : 1. Conclusions: These data indicate that this assay was effective for the specific detection of PrP determinants contained in bovine MBM extracts. Significance and Impact of the Study: To our knowledge, this is the first report detailing the detection of PrP determinants in bovine MBM. The assay could be applied to securing the safety of bovine MBM.