BACKGROUND/OBJECTIVES:To assess the performance of a food frequency questionnaire (FFQ) for estimating o-3, o-6 and trans fatty acid intake during pregnancy. Moreover, we determined whether the fatty acid composition of mature breast milk represents a valuable biomarker for fatty acid intake during pregnancy. SUBJECTS/METHODS: A prospective study in 41 pregnant women, aged 18-35 years, was conducted. Food intake during pregnancy was evaluated by three 24-h recalls (24 hR), and 2 FFQ. The fatty acid composition of mature breast milk was determined by gas chromatography. The method of triads and joint classification between quartiles of intake were applied. RESULTS: The FFQ was accurate for estimating docosahexanoic (DHA), linoleic and total o-6 fatty acids according to validity coefficients. Higher agreements (470%) into the same or adjacent quartiles between the dietary methods were found for a-linolenic, total o-3, linoleic and trans fatty acid intake. High validity coefficients for eicosapentanoic (EPA) and DHA acids of human milk were found (0.61 and 0.73, respectively), and the method was adequate for categorizing the intake of a-linolenic, total o-3 and trans fatty acids compared with FFQ estimates, and for arachidonic acid and trans fatty acids compared with food recall estimates, during pregnancy. CONCLUSIONS: The FFQ was an accurate tool for categorizing a-linolenic, total o-3 and trans fatty acid intake. According to the validity coefficients observed, the FFQ accurately estimated DHA, linoleic and total o-6 fatty acids and the composition of mature breast milk was shown to be a suitable biomarker for EPA and DHA fatty acid intake during pregnancy.