AbstractAn interleukin 3 (IL‐3)–dependent macrophage‐like cell line, 11‐1‐B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)–prelabeled cells showed that, after stimulation with the Ca2+ionophore A23187, the 11‐1‐B3 cells synthesized and released relatively large amounts of prostaglandin E2(PGE2) and leukotriene B4(LTB4) but not LTC4. In addition, 11‐1‐B3 cells showed Ca2+‐dependent and alkaline pH–optimal phospholipase A2(PLA2) activity that preferentially hydrolyzed cleavage ofsn‐2‐arachidonyl‐ but notsn‐2‐oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5–10 min resulted in five‐ to sevenfold increases in the membrane‐associated PLA2but activity in the cytosol was unchanged. This increase in membrane‐associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+directly activate membrane‐associated PLA2and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL‐3–dependent Mø‐like cells.