Abstract BAY X 1005 (R)-2-[4-(quinolin-2-yl-methoxy) phenyl]-2-cyclopentyl acetic acid has been demonstrated to be a potent inhibitor of leukotriene B 4 (LTB 4) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis in various in vitro systems. Using mainly human polymorphonuclear leukocytes (PMNL) this study elucidates the mechanism of inhibition of 5-lipoxygenase (5-LOX, EC 1.13. 11.34)-derived arachidonic acid metabolites by BAY X 1005. At concentrations of BAY X 1005 which almost totally inhibited the formation of 5-LOX-derived metabolites, both arachidonic acid release and platelet-activating factor synthesis were only modestly affected. This suggests that the inhibitory effect of BAY X 1005 is not due to a limitation of substrate availability for 5-LOX. Compared to the inhibition of leukotriene synthesis in intact human PMNL about 800-fold higher concentrations of BAY X 1005 were required to inhibit leukotriene formation in a cell-free system suggesting that the inhibitory effect of BAY X 1005 cannot be explained by a direct effect on 5-LOX. In an attempt to identify possible target proteins of BAY X 1005,[14 C] BAY X 1005 was used in binding studies under equilibrium conditions. The quantitative analysis of specific binding in intact human PMNL revealed two binding sites for BAY X 1005. Upon subcellular fractionation of these cells the BAY X 1005 high affinity binding site was localized in the microsomal fraction whereas the low affinity binding site was localized in the granule fraction. The K d for BAY X 1005 binding to the high affinity binding site (0.165 μmol L) was almost identical to the ic 50 value for inhibition of LTB 4 synthesis (0.22 μmol L). Furthermore, the ic 50 values for competition of BAY X 1005 binding at the high affinity binding site were almost identical to the ic 50 values for inhibition of LTB 4 synthesis in the case of BAY X 1005, 12 other structurally related quinoline derivatives and the reference compounds REV-5901, WY-50,295 and MK-886, but not in the case of the direct 5-LOX inhibitors A-64077 and AA-861. The analysis of BAY X 1005 binding in rat PMNL also revealed two binding sites. Whereas the low affinity binding site in rat PMNL exhibited a K d similar to the human, the rat high affinity binding site showed a 5.5-fold higher affinity for BAY X 1005 compared to the human. This correlates well with the 8.5-fold higher sensitivity of rat versus human PMNL concerning inhibition of LTB 4 synthesis. Competition experiments verified that this relationship holds true also for 12 other quinoline derivatives as well as for REV-5901, WY-50,295 and MK-886. Taken together, these results indicate a causal relationship between the binding of BAY X 1005 to the high affinity binding site and the inhibition of leukotriene synthesis in human and rat PMNL. In addition, the localization of this binding site in the microsomal fraction and the competition of BAY X 1005 binding by MK-886 suggest that the target protein of BAY X 1005 mediating leukotriene synthesis inhibition is identical to five lipoxygenase activating protein.