Primary cultures of [3H]arachidonic acid-prelabeled mouse peritoneal macrophages were stimulated with the physiologic agonists zymosan and Con A. The cells released a large quantity of labeled compounds into the extracellular medium. When the cells were preincubated for 10 min with PMA at concentrations from 10 to 1000 ng/ml, zymosan- and Con A-stimulated compound release was greatly enhanced. PMA also potentiated arachidonic acid release when cells were stimulated with the calcium ionophore A23187. However, under the same conditions, PMA treatment blocked agonist- and ionophore-mediated phosphoinositide hydrolysis in cells prelabeled with [3H]inositol. Thus, PMA treatment appears to have dissociated agonist-induced arachidonic acid liberation (index of phospholipase A2) from phosphoinositide hydrolysis (index of phospholipase C), suggesting that these two coupling processes can occur in a parallel and independent manner in mouse peritoneal macrophages. Furthermore, the ligand-induced arachidonic acid release from PMA-treated macrophages was shown to be directly dependent on the extracellular calcium concentration. Considering that in PMA-pretreated cells, receptor-mediated phosphoinositide breakdown and intracellular calcium mobilization were abolished, our data suggest that the extracellular calcium influx that takes place after receptor-ligand interaction may be a required event for arachidonic acid mobilization in mouse peritoneal macrophages.