Objective: To assess the relationship between habitual fish intake and fatty acid levels in serum as well as in the LDL fractions of serum phospholipids and cholesteryl esters. Design: Cross-sectional study. Setting: Cohort of Gipuzkoa (Basque Country, northern Spain) included in the European Prospective Investigation into Cancer and Nutrition (EPIC) project. Subjects: Random sample of 120 healthy volunteers of both sexes aged 35-65y, divided into various consumption groups according to daily fish intake. Methods: Data on habitual intake over the previous year was collected by trained interviewers by means of a computerized questionnaire based on the diet history method. Fasting venous blood samples were drawn and fatty acids were measured by gas-liquid chromatography. Results: Lean fish accounted for 78% of all fish consumption in the highest consumption group (> 115 g/day) and for 60% in the lowest (< 31 g/day). The mean concentrations of <omega>-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA, C20:5, omega -3), and docosahexaenoic acid (DHA, C22:6, omega -3) in serum and in the LDL fractions of serum phospholipids and cholesteryl esters increased significantly from the lowest to the highest fish consumption categories. Fish intake showed a statistically significant relationship with omega -3 PUFA, EPA and DHA in ser-um and in the LDL fractions of serum phospholipids and cholesteryl esters both in the simple linear regression analysis and in a multiple regression model adjusted by age, body mass index (BMI) and vegetable intake. Conclusions: Habitual fish intake is reflected in the content of EPA and DHA in serum and in the LDL phospholipid and cholesteryl esters fractions. The concentrations of very-long-chain omega -3 fatty acids are useful biomarkers for dietary fish intake, mainly lean fish.