Within an environmental risk assessment framework of a moderately contaminated estuary (the Sado, SW Portugal), the present work intended to detect endocrine disruption in a flatfish, Solea senegalensis Kaup, 1858, and its potential relationship to organic toxicants. Animals were collected from two distinct areas in the estuary (industrial and rural) and from an external reference area. Hepatic vitellogenin (VTG) levels, cytochrome P450 (CYP1A) induction, ethoxyresorufin-O-deethylase (EROD) activity plus gonad histology were analysed. Males and females were sexually immature and showed no significant evidence of degenerative pathologies. However, hepatic VTG concentrations in males from the industrial area were higher than Reference, even reaching levels comparable to females, indicating low but measurable oestrogenic effects caused by the complex contaminant mixture in estuarine sediments. These individuals also presented elevated CYP1A induction and EROD activity, which is consistent with contamination by organic toxicants such as PAHs and other aryl hydrocarbon receptor (Ahr) -mediated toxicants.