Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5 (S)-hydroperoxy-6-trans-8, 11, 14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA 4. 5-LOX is thought to receive arachidonic acid from the nuclear membrane–embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX–wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H (p) ETE (5-hydroxy-and 5-peroxy-6-trans-8, 11, 14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈ 60–70% 5-LOX-wt levels) but not of LTA 4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H (p) ETE at levels comparable to 5-LOX-wt but reduced LTA 4 hydrolysis products. Coexpression of FLAP partially restored LTA 4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.—Gerstmeier, J., Newcomer, ME, Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.