Identifying infected laying flocks is a critical component in efforts to prevent eggborne transmission of Salmonella enteritidis to humans. In the present study, egg yolk samples from experimentally infected chickens were tested for specific antibodies with a very rapid fluorescence polarization assay using tracers prepared from the O-polysaccharides of S. enteritidis and S. typhimurium and a conventional ELISA using an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with 106 or 10(8) cfu of S. enteritidis (phage type 13a) or with 10(8) cfu of S. typhimurium. Eggs were collected during five weekly postinoculation intervals. Both fluorescence polarization and ELISA detected the majority of hens infected with S. enteritidis at either dose level, although they also frequently cross-reacted with samples from hens infected with S. typhimurium. Fluorescence polarization with an S. typhimurium tracer was likewise able to consistently detect S. typhimurium infection but also tended to cross-react with samples from hens infected with S. enteritidis. Fluorescence polarization appears to offer a simple and rapid alternative to conventional serological methodology, although concerns about specificity may limit the usefulness of antibody testing data.