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Objectives: To evaluate the fatty acid composition of mature human milk of women living far from the coastal area of Brazil., Methods: Mature breast milk samples were obtained from 47 lactating women aged between 18 and 35 years, who delivered their babies at term and who exclusively or predominantly breastfed. Milk collection took place after the fifth week postpartum by hand expression. The fatty acid composition of the milk was determined by gas chromatography., Results: It was observed that the concentration of eicosapentaenoic acid (0.08%) was higher than that observed in previous studies in Brazil. However, the content of docosahexaenoic acid (0.09%) found in human milk was one of the lowest verified in the world. The content of trans fatty acids (2.05%) was similar to that reported in national studies previous to the mandatory declaration of this fatty acid content in food labels, suggesting that this measure had no effect on reducing the content of this fatty acid in the usual diet of women., Conclusions: Low levels of docosahexaenoic acid and high concentrations of trans fatty acids were observed in mature breast milk of women living far from the coastal area in Brazil., (Copyright © 2013 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.)