Raw oysters are primary vectors for Vibrio vulnificus infections, and a rapid detection method for V. vulnificus in raw oysters before distribution would be an indispensable tool for the seafood industry. One approach to improving the recovery and detection of V. vulnificus without sacrificing assay time is through the use of immunomagnetic separation (IMS). The aim of this study was to develop and optimize an IMS protocol using anti-H (antiflagellar) antibody for determining the level of V. vulnificus in phosphate-buffered saline (PBS) suspensions and spiked oyster homogenate. Six monoclonal antibodies were produced by immunizing mice at 2-week intervals by injection of 50 microg of purified V. vulnificus ATCC 27562 flagellin. Antibodies that exhibited high anti-H titers were coated onto Cowan I Staphylococcus aureus cells and sheep anti-mouse immunoglobulin G immunomagnetic beads. The two reagents were used to determine the species specificity of the selected antibodies, which positively identified and coagglutinated 70 isolates identified genetically as V. vulnificus and did not react with 40 Vibrio parahaemolyticus isolates or nine other Vibrio species. The IMS protocol was optimized for PBS and oyster homogenate spiked with three different strains of V. vulnificus. IMS with V. vulnificus-spiked PBS yielded binding of 19 to 57%, and IMS with spiked oyster homogenate carried out at two V. vulnificus levels exhibited binding of 25 to 57%. The IMS protocol for V. vulnificus could be used to concentrate and detect V. vulnificus in seawater and shellfish homogenate.