Leukotriene C₄ is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C₄ synthase (LTC₄S) participate in its biosynthesis. We report evidence that LTC₄S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC₄S. FLAP bound to the N-terminal part/first hydrophobic region of LTC₄S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC₄S. Fluorescent FLAP- and LTC₄S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC₄S co-localized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO translocated to the nuclear envelope allowing it to interact with FLAP and LTC₄S. Direct interaction of 5-LO and LTC₄S in ionophore-stimulated (but not un-stimulated) cells was demonstrated by BRET using GFP-5-LO and Rluc-LTC₄S.