The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes from arachidonic acid. In resting cells, 5-LO can accumulate in either the cytoplasm or the nucleoplasm and, upon cell stimulation, translocates to membranes to initiate leukotriene synthesis. Here, we used mutants of 5-LO with altered subcellular localization to assess the role that nuclear positioning plays in determining leukotriene B4 (LTB4) synthesis. Mutation of either a nuclear localization sequence or a phosphorylation site reduced LTB4 synthesis by 60%, in parallel with reduced nuclear localization of 5-LO. Mutation of both sites together or mutation of all three nuclear localization sequences on 5-LO inhibited LTB4 synthesis by 90% and abolished nuclear localization. Reduced LTB4 generation in mutants could not be attributed to differences in 5-LO amount, enzymatic activity, or membrane association. Instead, 5-LO within the nucleus acts at a different site, the nuclear envelope, than does cytosolic 5-LO, which acts at cytoplasmic and perinuclear membranes. The significance of this difference was suggested by evidence that exogenously derived arachidonic acid colocalized with activated nuclear 5-LO. These results unequivocally demonstrate that the positioning of 5-LO within the nucleus of resting cells is a powerful determinant of the capacity to generate LTB4 upon subsequent activation.