A protective association between breastfeeding and the development of bronchial asthma has been demonstrated. However, a mechanism remains unclear. FA present in human milk but rare in infant formula have been associated with marked immunological modulation as well as some indications of protection from asthma development. We examined the effect of in vitro manipulation of membrane phospholipid on the production of cytokines and prostaglandin (PG)E2 by respiratory epithelial cells (A549) in response to stimulation by mast cell mediators of allergic disease [histamine, tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-5]. DHA and CLA significantly decreased the production of IL-8 in response to stimulation by TNF-α [2907 ± 970 (DHA) and 6471 ± 1203 (CLA) vs. 12,287 ± 2309 (control) pg/mL; P ≤ 0.05, mean ± SEM], whereas both EPA and DHA reduced histamine-stimulated RANTES (regulation on activation, T cell-expressed and -secreted) production [2314 ± 861 (EPA) and 877 ± 326 (DHA) vs. 8526 ± 1118 (control) pg/mL; P ≤ 0.03]. PGE2 released in response to histamine was decreased by n-3 [1305 ± 399 (α-linolenic acid), 406 ± 73 (EPA), and 265 ± 32 (DHA) vs. 9324 ± 3672 (control) pg/mL; P ≤ 0.05] and increased by n-6 [18,843 ± 4439 (arachidonic acid) vs. 9324 ± 3672 (control) pg/mL; P = 0.02], with CLA producing a decrease of the same magnitude as DHA [553 ± 126 (CLA) vs. 9324 ± 3672 (control) pg/mL; P = 0.03]. This study demonstrates the potential for immunological manipulation of the respiratory epithelium by FA in situ during allergic responses and suggests that further investigation into FA intervention in infants via human milk or supplemented infant formula, to prevent the development of allergic disease, may be worthwhile.