The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC. Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method. The present system allows for the efficient and simultaneous determination of various DEC pathotypes. This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.