5‐Lipoxygenase (5‐LO) and its activating protein (FLAP) are both required for cellular leukotriene (LT) synthesis, with 5‐LO catalyzing both the synthesis of (5S)‐5‐hydroperoxy 6,8,11,14‐eicosatetraenoic acid (5‐HPETE) from arachidonic acid and the subsequent synthesis of LTA₄ from 5‐HPETE. We have previously expressed both human 5‐LO and human FLAP to high levels in Spodoptera frugiperda (Sf9) insect cells, using recombinant baculoviruses. To study the mechanism by which FLAP activates 5‐LO, we compared cellular 5‐LO activity in Sf9 cells expressing this enzyme to that in Sf9 cells coexpressing FLAP and 5‐LO. In this system, FLAP stimulates the utilization of arachidonic acid by 5‐LO as a substrate, and increases the efficiency with which 5‐LO converts 5‐HPETE to LTA₄. LT synthesis in cells coexpressing FLAP and 5‐LO is inhibited by 3‐[1‐p‐chlorophenyl)‐5‐isopropyl‐3‐tert‐butylthio‐1H‐indol‐2‐yl]‐2,2‐dimethyl‐propanoic acid (MK‐886), an LT biosynthesis inhibitor which specifically binds to FLAP. These studies in Sf9 cells, together with our recent demonstration that FLAP specifically binds arachidonic acid, suggests that FLAP activates 5‐LO by acting as an arachidonic acid transfer protein.