The biochemical responses in muscle, such as the enzymatic activities of acetylcholinesterase, lactate dehydrogenase and isocitrate dehydrogenase, were studied in sole (Solea senegalensis) collected in Huelva estuary (SW Spain), in the vicinity of a petrochemical and mining industry. The sampling sites showed different type and degree of pollution. The results demonstrated significant differences in muscle activities of AChE and IDH in Odiel and Tinto Rivers compared to control fish. LDH activity did not show any difference between sampling sites. Significant correlations were established between some biomarkers and heavy metals: AChE was correlated with Pb, Cd and Cu concentrations in water; IDH activity was correlated with Cd and Cu concentrations in water and As, Pb and Cd concentrations in sediments; LDH activity was correlated with As and Zn concentration in water and Cd concentration in sediment. Only one correlation was established between the biomarkers analysed and the concentrations of PAHs: benzo(b)fluoranthene concentration in sediment and IDH.