5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.