The structure-activity relationship of 18-carbon fatty acids (C18FAs) on human neutrophil functions and their underlying mechanism were investigated. C18unsaturated (U)FAs potently inhibited superoxide anion production, elastase release, and Ca2+mobilization at concentrations of <10 μM in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-activated human neutrophils. However, neither saturated FA nor esterified UFAs inhibited these neutrophil functions. The inhibitory potencies of C18UFAs decreased in the following order: C18:1 > C18:2 > C18:3 > C18:4. Notably, the potency of attenuating Ca2+mobilization was closely correlated with decreasing cellular responses. The inhibitions of Ca2+mobilization by C18UFAs were not altered in a Ca2+-containing Na+-deprived medium. Significantly, C18UFAs increased the activities of plasma membrane Ca2+-ATPase (PMCA) in neutrophils and isolated cell membranes. In contrast, C18UFAs failed to alter either the cAMP level or phosphodiesterase activity. Moreover, C18UFAs did not reduce extracellular Ba2+entry in FMLP- and thapsigargin-activated neutrophils. In summary, the inhibition of neutrophil functions by C18UFAs is attributed to the blockade of Ca2+mobilization through modulation of PMCA. We also suggest that both the free carboxy group and the number of double bonds of the C18UFA structure are critical to providing the potent anti-inflammatory properties in human neutrophils.