The intracellular distribution of the enzyme 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) in resting and ionophore-activated human leukocytes has been determined using immuno-electronmicroscopic labeling of ultrathin frozen sections and subcellular fractionation techniques. 5-LO is a 78-kD protein that catalyzes the conversion of arachidonic acid to leukotrienes. FLAP is an 18-kD membrane bound protein that is essential for leukotriene synthesis in cells. In response to ionophore stimulation, 5-LO translocates from a soluble to a sedimentable fraction of cell homogenates. In activated leukocytes, both FLAP and 5-LO were localized in the lumen of the nuclear envelope. Neither protein could be detected in any other cell compartment or along the plasma membrane. In resting cells, the FLAP distribution was identical to that observed in activated cells. In addition, subcellular fractionation techniques showed > 83% of immunoblot-detectable FLAP protein and approximately 64% of the FLAP ligand binding activity was found in the nuclear membrane fraction. A fractionation control demonstrated that a plasma membrane marker, detected by a monoclonal antibody PMN13F6, was not detectable in the nuclear membrane fraction. In contrast to FLAP, 5-LO in resting cells could not be visualized along the nuclear envelope. Except for weak labeling of the euchromatin region of the nucleus, 5-LO could not be readily detected in any other cellular compartment. These results demonstrate that the nuclear envelope is the intracellular site at which 5-LO and FLAP act to metabolize arachidonic acid, and that ionophore activation of neutrophils and monocytes results in the translocation of 5-LO from a nonsedimentable location to the nuclear envelope.