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Background: PUFAs may influence the risk of asthma; however, long-term prospective studies including objective biomarkers of PUFA intake are lacking., Objectives: The objective was to investigate the role of dietary intake and plasma concentrations of n-3 and n-6 (ω-3 and ω-6) PUFAs in childhood and adolescence for the development of asthma and lung function up to young adulthood., Methods: The study included participants from the Swedish prospective birth cohort BAMSE. Dietary intake of PUFAs was calculated from FFQs (n = 1992) and plasma proportions of PUFAs were measured in phospholipids (n = 831). We analyzed the n-3 PUFA α-linolenic acid (ALA; 18:3n-3); the sum of very-long-chain (VLC) n-3 PUFAs: EPA (20:5n-3), DHA (22:6n-3), and docosapentaenoic acid (22:5n-3); and the n-6 PUFAs linoleic acid (LA; 18:2n-6) and arachidonic acid (AA; 20:4n-6). Asthma was assessed by questionnaires at 8, 16, and 24 y and lung function was measured by spirometry at 24 y., Results: A high (≥median) self-reported dietary intake of LA at 8 y and AA at 16 y was associated with increased risk of prevalent asthma at 24 y (OR: 1.41; 95% CI: 1.10, 1.82 and OR: 1.32; 95% CI: 1.02, 1.70, respectively). In contrast, plasma proportions of ALA, ∑VLC n-3 PUFAs, and AA at 8 y, as well as LA at 16 y, were inversely associated with prevalent asthma at 24 y (e.g., OR: 0.55; 95% CI: 0.38, 0.81 for ∑VLC n-3 PUFAs). No consistent associations were observed with lung function., Conclusions: High dietary intake of certain n-6 PUFAs in childhood or adolescence may be associated with increased risk of asthma up to young adulthood, whereas dietary biomarkers of certain n-3 and n-6 PUFAs in plasma may be associated with decreased risk. Thus, the role of diet compared with altered metabolism of PUFAs needs further investigation to improve dietary preventive strategies for asthma., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)