Leukotrienes (LT) are potent lipid mediators derived from arachidonic acid (AA) via the 5-LO pathway [1]. AA is converted to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE), which in turn is metabolized to LTA4. In the cell, 5-LO activity depends on a crucial translocation from the soluble compartment to the nuclear membrane-bound 5-lipoxygenase-activating protein (FLAP) to achieve endogenously released AA as substrate for LT biosynthesis [2]. Although 5-LO and LT biosynthesis is known for more than 35 years, a role in the development of cardiovascular diseases (CVD), cancer, and Alzheimer`s disease besides chronic allergic diseases like asthma has only recently been discovered [3]. Inspired by the therapeutic potential, BRP-7 was identified as a new class of FLAP inhibitors with high efficacy to inhibit LT biosynthesis in vitro and in vivo. Additionally, a powerful mammalian stable expression system of 5-LO and FLAP in HEK293 cells was established to clearly determine putative FLAP inhibitors and study FLAPs undisputed significance for cellular LT biosynthesis. FLAP increases cellular 5-LO product formation by enhancing the LTA4 synthase activity, and supports 5-LO membrane accumulation. For the first time, we provide strong evidence for an effective in situ interaction of native 5-LO and FLAP at the nuclear membrane in primary human leukocytes and stable transfected HEK293 cells by proximity ligation assay (PLA). 5-LO/FLAP interaction occurs delayed to 5-LO activity and translocation. FLAP antagonists prevent the 5-LO/FLAP interaction, and AA and/or 5-HPETE function as regulating molecules for the 5-LO/FLAP complex assembly.