The distribution of cytosolic phospholipase A2 (cPLA2), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA2 and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A23187, both proteins accumulated primarily in nuclei-containing fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions. Further resolution of nuclear fractions revealed that 5-lipoxygenase and cPLA2 were localized in a fraction that contained nuclear membranes. In comparison, 5-LAP was localized to the nuclear-membrane fraction of resting and activated neutrophils, as determined by immunoblotting and photoaffinity labeling. In agreement with the immunoblot data, A23187 stimulation markedly enhanced 5-lipoxygenase enzymatic activity in the nuclear-membrane fraction, which was accompanied by decreased cytosolic 5-lipoxygenase activity. Similarly, neutrophil activation caused increased phosphorylation of cPLA2, a process that is known to result in enhanced catalytic activity. Our data demonstrate that in activated human neutrophils, the key proteins involved in leukotriene synthesis colocalize at the nuclear membrane, in a catalytically active state.