Phosphatidate (PA) is synthesized by a variety of cells in response to physiological agonists. Addition of PA vesicles to [3H]arachidonic acid (AA)-labeled macrophages was found to induce the release of radiolabel in a dose- and time-dependent manner. This effect correlated with the uptake of PA by the macrophages and appeared to be attributable to PA itself and not to a PA metabolite. In parallel with AA release, PA induced a rapid increase in lysophosphatidylcholine in cells prelabeled with [14C]glycerol. Down-regulation of protein kinase C by long term exposure of the cells to phorbol myristate acetate or cell treatment with the protein kinase C inhibitor staurosporine did not affect the PA response. Also, removal of external calcium or cell treatment with the calmodulin antagonist trifluoperazine did not affect PA-induced AA release, while inhibiting the responses to zymosan, phorbol 12-myristate 13-acetate, and ionophore A23187. PA-induced AA release was not affected by intracellular calcium depletion by treatment with quin2/AM in the presence of EGTA. When assayed toward an AA-containing substrate, PA was able to enhance phospholipase A2 activity from cell homogenates in the absence of calcium. The dose dependence and magnitude of the PA effect correlated with those observed for PA-induced AA release in whole cells. Inclusion of ATP in the assay mixture did not affect the activity of the PA-stimulated phospholipase A2. These findings suggest a role for PA in the cascade of events leading to AA release in macrophages through Ca(2+)-independent stimulation of an AA-selective phospholipase A2.