Assessing toxicity of contaminated estuarine sediments poses a challenge to ecotoxicologists due to the complex geochemical nature of sediments and to the combination of multiple classes of toxicants. Juvenile Senegalese soles were exposed for 14 days in the laboratory and in situ (field) to sediments from three sites (a reference plus two contaminated) of a Portuguese estuary. Sediment characterization confirmed the combination of metals, polycyclic aromatic hydrocarbons and organochlorines in the two contaminated sediments. Changes in liver cytosolic protein regulation patterns were determined by a combination of two-dimensional electrophoresis with de novo sequencing by tandem mass spectrometry. From the forty-one cytosolic proteins found to be deregulated, nineteen were able to be identified, taking part in multiple cellular processes such as anti-oxidative defence, energy production, proteolysis and contaminant catabolism (especially oxidoreductase enzymes). Besides a clear distinction between animals exposed to the reference and contaminated sediments, differences were also observed between laboratory- and in situ-tested fish. Soles exposed in the laboratory to the contaminated sediments failed to induce, or even markedly down-regulated, many proteins, with the exception of a peroxiredoxin (an anti-oxidant enzyme) and a few others, when compared to reference fish. In situ exposure to the contaminated sediments revealed significant up-regulation of basal metabolism-related enzymes, comparatively to the reference condition. Down-regulation of basal metabolism enzymes, related to energy production and gene transcription, in fish exposed in the laboratory to the contaminated sediments, may be linked to sediment-bound contaminants and likely compromised the organisms' ability to deploy adequate responses against insult.