The opportunistic food pathogen Listeria monocytogenes is of great concern to the food industry and its rapid detection is of major importance. This paper describes the detection of L. monocytogenes with a polyclonal antibody by means of a new subtractive inhibition assay using a BIAcore 3000 biosensor. Incubating L. monocytogenes cells and antibody for a short period of time, followed by subsequent separation of free unbound antibody with a stepwise centrifugation process, allowed the detection of 1 x 10(5) L. monocytogenes cells/ml in less than 30 min. Free antibody was passed over an anti-Fab ligand-coated sensor chip surface with the generated response being inversely proportional to the inhibiting cell concentration. The method was simple, rapid and needed minimum sample preparation. This assay format has the potential for the quick and sensitive detection of pathogens with limited sample handling and preparation.