A bioassay using the YCM3 recombinant yeast strain was utilised to investigate the presence of dioxin-like compounds that activate the aryl hydrocarbon receptor (AhR) in treated sewage effluents. AhR ligand activity was detected in the concentrated extracts of effluent samples collected in March, June and October 2004 from Kyoto city, Japan. HPLC fractionation was carried out using C18 reversed-phase columns, and possible AhR ligands were further isolated and purified. By using LC/MS/MS, one weak AhR ligand was identified to be rhodamine B base, a fluoran dye. In addition, two other coloured ligands were postulated to be disperse anthraquinone dyes or their metabolites because of their UV spectra and HPLC retention times. The AhR-binding affinities of 12 commercial dyes with different chemical structures were also studied. Among the dyes tested, hydrophobic anthraquinone dyes exhibited higher AhR ligand activity, but azo dyes or hydrophilic acid dyes showed no or very low AhR ligand activity. Rhodamine B base and disperse anthraquinone dyes were suggested to be potential xenobiotic AhR ligands. Future research regarding their contamination in aquatic environments and toxicological information is necessary.