The results of the present study are based on enzyme biomarker measurements in flounder (Platichthys flesus), a flatfish species that is widely distributed in Baltic coastal waters. The fish were collected from known pollution gradients and from reference areas regarded as relatively free of anthropogenic input. Muscular cholinesterases (ChEs), hepatic glutathione S-transferase (GST), and hepatic ethoxyresorufin-O-deethylase (EROD) activities were measured in each sampled specimen of flounder. Generalized linear models (GLM) were used to analyze the dependence of the enzyme activity on sampling year and area as well as on the biological parameters of the fish. Statistically significant differences in the activities of the measured biomarkers were observed between reference and contaminated sites. ChEs and GST activities differed with gender. The results of this study suggest that the location and year of sampling have a significant impact on the activity of all the measured biomarkers.