The aim of this study was to develop a fluorescence polarization immunoassay (FPIA) that is based on the change in fluorescence polarization of fluorescently labeled small antigen when bound by a specific antibody, for use as a screening test for zearalenone (ZEN) in cereals and their products. Syntheses of fluorescein-labeled ZEN tracers containing three linkers of different lengths (2, 3 and 6-carbon bridge), ethylenediamine, 1,2-diaminopropane and hexamethylenediamine, were explored and their binding response with ZEN-specific antibody was evaluated. A fluoresceinthiocarbamyl hexamethylenediamine-labeled ZEN conjugate (ZEN-HMDF), which contain a 6-carbon bridge, was found to be the most sensitive FPIA for detection of ZEN. When tested on corn matrix the FPIA using the ZEN-HMDF tracer showed a detection range for ZEN of 150-1000 microg kg(-1) with a detection limit of 137 microg kg(-1), and required less than 2 min per sample to carry out, excluding extraction time. The average recovery from spiked corn samples was 106.4+/-12.5%. Comparative analyses of 70 naturally occurring cereals and their products using FPIA, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) showed that the coefficients of determination (r(2)) between FPIA and ELISA, and between FPIA and HPLC were 0.76 and 0.72, respectively. These results suggest that the ZEN-HMDF tracer is suitable for FPIA, which has potential as a screening tool for ZEN in grains without the need for a complicated clean-up procedure.