5‐Lipoxygenase (5‐LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5‐LO translocates to the nuclear membrane where arachidonic acid is transferred by 5‐LO‐activating protein (FLAP) to 5‐LO for metabolism. Although previous data indicate association of 5‐LO with FLAP, the in situ assembly of native 5‐LO/FLAP complexes remains elusive. Here, we show time‐resolved 5‐LO/FLAP colocalization by immunofluorescence microscopy and in situ 5‐LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca²⁺‐ionophore A23187‐activated human monocytes and neutrophils in relation to 5‐LO activity. Although 5‐LO translocation and product formation is completed within 1.5‐3 min, 5‐LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5‐LO protein, neutrophils produce 3‐5 times higher levels of 5‐LO products due to prolonged activity, accompanied by delayed 5‐LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5‐LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 μM) disrupt the complex. We conclude that FLAP may regulate 5‐LO activity in 2 ways: first by inducing an initial flexible association for efficient 5‐LO product synthesis, followed by the formation of a tight 5‐LO/FLAP complex that terminates 5‐LO activity.—Gerstmeier, J., Weinigel, C., Rummler, S., Rådmark, O., Werz, O., Garscha, U. Time‐resolved in situ assembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes. FASEB J. 30, 276‐285 (2016). www.fasebj.org