The aim of the study was to examine temporal (monthly) and spatial changes in the activities of a battery of biomarkers and evaluate the influence of gender and environmental factors on those activities. Abiotic factors not correlated with chemical pollution-water temperature, salinity, and oxygen concentration-were evaluated. The activities of the following biomarkers-muscular ChEs (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)), hepatic ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST) and catalase (CAT), and gross indices-condition factor (CF), hepatosomatic index (HSI), and gonadosomatic index (GSI)-were measured in individual flounder Platichthys flesus. Female flounder were collected once a month from April 2003 to April 2004 at Sobieszewo (Gulf of Gdańsk). In December 2003 flounder of both sexes from nine stations in the Baltic Proper were caught and tested. Strong monthly (attributed mainly to spawning, and less-to pollution) and geographical (attributed to pollution) variations in biomarker activities, as well as gender differences, were demonstrated. Correlations between the enzymes with the abiotic properties of the environment were less important.