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Abstract: This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) α in the Lake Biwa–Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARα agonist contamination in the river basins. RARα agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6ng-atRA/L and 23.5ng-atRA/L being observed in Lake Biwa–Yodo River and Ina River, respectively. The results indicated that RARα agonists are always present and widespread in the rivers. Comparative investigation of RARα and estrogen receptor α agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARα agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARα agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARα agonists in the rivers. Although a trial conducted to identify RARα agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography–mass spectrometry analysis revealed that the major causative contaminants responsible for the RARα agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARα agonists with high activity in the rivers. [Copyright &y& Elsevier]