We previously reported that zymosan-stimulated, protein kinase C (PKC)-dependent arachidonic acid liberation occurs with association of Ca 2 + -independent phospholipase A 2 (iPLA 2 ) with the membranes of macrophage-like P388D 1 cells. In the present study, the possible involvement of PKC isoforms (α, β, δ, and ) on the increase in iPLA 2 was examined. Stimulation of P388D 1 cells with zymosan induced increases in iPLA 2 activity and protein in the membranes and liberation of arachidonic acid. In the stimulated cells, PKCα, PKCδ, and PKC , but not PKCβ, were increased in the membranes. The zymosan-induced increase in iPLA 2 activity was suppressed by pretreatment with 4β-phorbol 12-myristate 13-acetate for 10hr, by which PKCα and PKCδ, but not PKCβ and PKC , were depleted, and by Go6976, a PKCα inhibitor, but not rottlerin, a PKCδ inhibitor. The zymosan-induced release of arachidonic acid was also reduced by the PKC depletion and Go6976. However, stimulation with 4β-phorbol 12-myristate 13-acetate alone did not increase iPLA 2 activity in the membranes. Furthermore, the depletion of intracellular Ca 2 + also impaired the zymosan-induced increase in iPLA 2 activity in the membranes. However, no increase in iPLA 2 activity was observed upon stimulation with Ca 2 + -mobilizing agents (ionomycin or thapsigargin). Cytochalasin D, an inhibitor of actin polymerization, suppressed the zymosan-induced increases in iPLA 2 activity and protein in the membranes and the release of arachidonic acid. These results suggest that zymosan stimulates an increase in iPLA 2 in the membranes of P388D 1 cells probably through activation of PKCα in concert with cytochalasin D-sensitive events.