Objective: To investigate the fatty acid composition of mature human milk in Western Iran with special focus on trans fatty acids. Design: Observational study. Methods: Milk samples were collected from 52 lactating mothers aging 19-39 y, from Western Iran. Subjects were asked to complete a diet questionnaire. Milk fatty acids were measured as 2-nitrophenylhydrazide derivatives by high-performance liquid chromatography. Results: Saturated fatty acids were the main fraction of human milk (41.3%). Medium-chain fatty acids (C8:0-C14:0) constituted 24%, oleic acid (C18:1o9) accounted for 30.9% and elaidic acid (C18:1T), the trans isomer of oleic acid, comprised 11.3% of the total milk fatty acids. Linoleic (C18:2o6) and linolenic (C18:3o3) acid contents were 13.8 and 1.1%, respectively. The level of the polyunsaturated fatty acids was 1.4% for arachidonic (C20:4o6) and 0.2% for eicosapentaenoic (C20:5o3) acid. Conclusions: The milk from Iranian lactating mothers, as compared to that from the American or European mothers, contained high levels of medium-chain and trans fatty acids. This difference may be attributed to the maternal diet with low animal protein and animal fat but with high carbohydrate and partially hydrogenated vegetable oils that carry large amounts of trans fatty acids. As the detrimental effects of trans fatty acids on blood lipids and cardiovascular diseases have been emphasized in the literature, a reduction of trans fatty acid content in the diet of Iranian mothers is suggested.