Stimulation of resident peritoneal macrophages resulted in release of arachidonic acid (AA) from phospholipids. This AA release is believed to occur as a result of the activation of phospholipases, usually by a phospholipase A2 (PLA2). The purpose of this study was to elucidate the role of cytosolic calcium ion concentration ([Ca2+]i) in the modulation of [3H]AA mobilization in peritoneal macrophages. [3H]AA release induced by ionophore A23187, opsonized zymosan, or 4 beta-phorbol 12-myristate acetate (PMA) occurred in the absence of extracellular calcium. Studies in fura-2/AM-loaded cells showed that zymosan and PMA did not increase [Ca2+]i significantly, whereas A23187 induced PLA2 activity translocation up to plasmatic membrane. Thapsigargin, an inhibitor of endomembrane Ca(2+)-ATPase, induced a rise in [Ca2+]i when cells were incubated in a Ca2+ medium. However, thapsigargin was not an effective stimulator of the translocation of PLA2 activity and [3H]AA release. These data indicate that changes in [Ca2+]i were not sufficient to elicit [3H]AA mobilization; this process seems tightly modulated by phosphorylation-dependent mechanisms in the presence of low [Ca2+]i.