A procedure for simultaneous quantification of Campylobacter and Salmonella spp. in poultry skin rinse fluids by a flotation and real-time multiplex PCR method is described. Flotation of the target organisms in a discontinuous density gradient separated them from background microflora, particles from poultry skin, dead target cells and PCR inhibitors. Variation of the buoyant density between 1.052 to 1.106 g/ml was measured at different times for various Salmonella strains grown over a period of 4 weeks. This, and the results from earlier studies on the buoyant densities of Campylobacter spp., which were between 1.065 and 1.109 g/ml, led to design of an optimal discontinuous flotation method with three density layers, of 1.048, 1.109 and approximately 1.200 g/ml. This method preceded a real-time multiplex PCR assay using hybridization probes. The specificity of the PCR assay was confirmed on 73 target and non-target strains, and target organisms were detected at the level of one genome per PCR. Results obtained with the combined flotation and real-time multiplex PCR method showed that quantification in rinse fluids was possible down to 3.0+/-0.3 x 10(3) CFU/ml in the presence of other microorganisms at numbers up to 10(9) CFU/ml.