The in vitro actions were investigated of LY293111, a potent and selective leukotriene B₄ (LTB₄) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC₅₀ for inhibiting [³H]LTB₄binding to human neutrophils was 17.6 ± 4.8 nM. LY293111 inhibited LTB₄-induced human neutrophil aggregation (IC₅₀ = 32 ± 5 nM), luminol-dependent chemiluminescence (IC₅₀ = 20 ± 2 nM), chemotaxis (IC₅₀ = 6.3 ± 1.7 nM), and superoxide production by adherent cells (IC₅₀ = 0.5 nM). Corresponding responses induced byN-formyl-l-methionyl-l-leucyl-l-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB₄ binding to guinea pig tissues and subsequent activation were also inhibited. The K _i for inhibition of [³H]LTB₄ binding to lung membranes was 7.1 ± 0.8 nM; IC₅₀ for preventing binding of [³H]LTB₄ to spleen membranes was 65 nM. The compound inhibited LTB₄-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB₄ concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB₄ and thromboxane B₂ by plasma-depleted human blood stimulated withN-formyl-l-methionyl-l-leucyl-l-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB₄ by A23187-activated whole blood and conversion of arachidonic acid to LTB₄ by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB₄ receptor antagonist with much improved potency in a variety of functional assay systems.