We have studied the translocation of cytosolic phospholipase A₂ (cPLA₂) to nuclei in macrophages stimulated with receptor-recognized forms of α₂-macroglobulin (α₂M*). Translocation of phosphorylated cPLA₂ to nuclei was determined by immunoprecipitation of cPLA₂ in ³²P_i-labeled cells. The identity of cPLA₂ was established by comparing its mobility on gels with an authentic cPLA₂ standard. cPLA₂ activity was quantified by measuring the release of [¹⁴C]arachidonic acid from the substrate 1-palmitoyl-2-[1-¹⁴C]arachidonyl-sn-glycerophosphatidylcholine. α₂M* caused a two- to threefold increase in cPLA₂ phosphorylation and its translocation to nuclei. The p38 MAPK inhibitor SB203580, PKC inhibitor chelerythrin, or depletion of intracellular Ca²⁺ profoundly decreased cPLA₂ activity in nuclei isolated from agonist-stimulated cells. The requirement for Ca²⁺, PKC, and p38 MAPK activation appears to be of major importance for nuclear cPLA₂ activity. In contrast to cellular cPLA₂ activity, nuclear cPLA₂ activity was not inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃) in agonist-stimulated cells. It is concluded that the association of cPLA₂ with nuclear membranes in agonist-stimulated cells modifies the activity and the sensitivity of the enzyme to inhibition by AACOCF₃ in this phospholipid environment.