Receptor-stimulated arachidonic acid (AA) mobilization in P388D1 macrophages consists of a transient phase in which AA accumulates in the cell and a sustained phase in which AA accumulates in the incubation medium. We have shown previously that a secretory group II phospholipase A2 (sPLA2) is the enzyme responsible for most of the AA released to the incubation medium. By using selective inhibitors for each of the PLA2s present in P388D1 macrophages, we demonstrate herein that the cytosolic group IV PLA2 (cPLA2) mediates accumulation of cell-associated AA during the early steps of P388D1 cell activation. The contribution of both cPLA2 and sPLA2 to AA release can be distinguished on the basis of the different spatial and temporal characteristics of activation and substrate preferences of the two phospholipase A2s (PLA2s). Furthermore, the results suggest the possibility that a functionally active cPLA2 may be necessary for sPLA2 to act. cPLA2 action precedes that of sPLA2, and overcoming cPLA2 inhibition by artificially increasing intracellular free AA levels restores extracellular AA release. Although this suggests cross-talk between cPLA2 and sPLA2, selective inhibition of one other PLA2 present in these cells, namely the Ca2+-independent PLA2, does not block, but instead enhances receptor-coupled AA release. These data indicate that Ca2+-independent PLA2 does not mediate AA mobilization in P388D1 macrophages. Collectively, the results of this work suggest that each of the PLA2s present in P388D1 macrophages serves a distinct role in cell activation and signal transduction.