Biological-effects monitoring in estuarine environments is complex as a result of strong gradients and fluctuations in salinity and other environmental conditions, which may influence contaminant bioavailability and the physiology and metabolism of the organisms. To select the most robust and reliable biological-effect methods for monitoring and assessment programmes, a large-scale field study was conducted in two estuarine transects in the Netherlands. The locations ranged from heavily polluted harbour areas (the ports of Rotterdam and Amsterdam) to cleaner coastal and freshwater sites. Assessment methods used included a variety of biomarkers in flounder (Platichthys flesus) and a range of in vitro (sediment extracts) and in vivo bioassays. Multivariate statistical analysis was applied to investigate correlations and relationships between various biological effects and contaminant levels in flounder liver or sediments. Several biological methods seemed to be too much affected by salinity differences for routine use in estuaries. The most discriminative biomarkers in the study were hepatic metallothionein content and biliary 1-OH pyrene in fish. Mechanism-based in vitro assays DR-CALUX and ER-CALUX applied to sediment extracts for screening of potential toxicity were much more responsive than in vivo bioassays with macro-invertebrates using survival as an endpoint. © 2009 International Council for the Exploration of the Sea. Published by Oxford Journals. All rights reserved.