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 -65 y, 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 o-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA, C20:5, o-3), and docosahexaenoic acid (DHA, C22:6, o-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 o-3 PUFA, EPA and DHA in serum 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 o-3 fatty acids are useful biomarkers for dietary fish intake, mainly lean fish. Sponsorship: Europe Against Cancer Programme of the European Union (agreement SOC 97 200302 05F02); 'Fondo de Investigaciones Sanitarias', Spanish Ministry of Health (FIS grant 99=0024-05); Government of the Basque Country; and 'Fundación Científica de la Asociación Española contra el Cáncer'. Descriptors: fish intake; fish oil; n-3 fatty acids; biological markers; eicosapentaenoic acid; docosahexaenoic acid