RATIONALE: Exposure to air pollution is associated with adverse respiratory effects. Omega-3 polyunsaturated fatty acids (n-3 FA) appear to attenuate the health effects to air pollution. OBJECTIVE: This panel study evaluated whether n-3 FA intake and blood levels of omega-6 polyunsaturated fatty acids (n-6 FA) can modulate the associations between respiratory effects and short-term exposure to ambient air pollution in healthy adults. METHODS: Sixty-two healthy adults were enrolled into either high or low n-3 groups based on n-3 FA intake and erythrocytes n-3 FA concentrations. Low and high n-6 groups were dichotomized on blood n-6 FA levels. Participants underwent 3-5 testing sessions separated by at least seven days. At each session, FVC, FEV1, plasma markers of inflammation (IL-6) and oxidative stress (ox-LDL) were measured. Associations between ambient ozone and fine particulate matter (PM2.5) levels and lung function and blood markers were assessed using mixed-effects models stratified by fatty acids levels. RESULTS: Average concentrations of ozone (40.8±11.1 ppb) and PM2.5 (10.2±4.1 µg/m3) were below national ambient air quality standards during the study period. FVC was positively associated with ozone at lag0 in the high n-3 group while the association was null in the low n-3 group [for an IQR increase in ozone, 1.8%(95% CI:0.5-3.2) vs. 0.0%(95% CI:-1.4-1.5)]; however, the association shifted to negative at lag4 [-1.9%(95% CI:-3.2- -0.5) vs. 0.2%(95% CI:-1.2-1.5)] and lag5 [-1.2%(95% CI:-2.4-0.0) vs. 0.9%(-0.4-2.3)]. A similar pattern was observed in the low n-6 group compared to the high n-6 group [lag0:1.7%(95% CI:0.3-3.0) vs. 0.5%(95% CI:-0.9-2.0) and lag4:-1.4%(95% CI:-2.8-0.0) vs. -0.5%(95% CI:-1.8-0.9)]. The associations between FEV1 and ozone and between FVC and PM2.5 also followed a similar pattern. Elevated ozone levels were associated with an immediate decrease in ox-LDL in the high n-3 group atlag0 [-12.3%(95% CI:-24.8-0.1)] while no change in the low n-3 group [-7.5%(95% CI: -21.4-6.5)], and a delayed increase in IL-6 in the high n-3 group compared with the low n-3 group [lag4: 66.9%(95% CI:27.9-106.0) vs. 8.9%(95% CI:-31.8-49.6), lag5: 58.2%(95% CI:22.4-94.1) vs. -7.4%(95% CI:-48.8-34.0), and lag6: 45.8%(95% CI:8.7-82.9) vs. -8.5%(95% CI:-49.7-32.6)]. CONCLUSIONS: We observed lag-dependent associations between short-term ambient air pollutants and lung function that were differentially modulated by n-3 and n-6 FAs, suggesting that n-3 and n-6 FAs counteract the respiratory response to low levels of ambient air pollution in healthy adults. Clinical trial registered with ClinicalTrials.gov (NCT02921048).