Stimulation of macrophages with IgG2 immune complexes induced dose-dependently the O₂^- generation and the release of arachidonic acid and its metabolites. This Fc_γR-mediated O₂^- generation was inhibited by a phospholipase A₂ inhibitor, 4-p-bromophenacyl bromide (4-pBPB), in parallel to the dose-dependent inhibition of arachidonic acid release. The main arachidonic acid metabolites released were shown to be prostaglandin E₂ and thromboxane B₂ and blocking of the production of these metabolites by indomethacin did not inhibit the O₂^- generation. Inhibition of the Fc_γR-mediated O₂^- generation and the arachidonic acid release by the C-kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), was less intense than by 4-pBPB. These results support the previously proposed hypothesis that arachidonic acid acts as an intracellular activator of the Fc_γR-mediated O₂^- generation in macrophages. Although the C-kinase activation may also contribute to the activation of the O₂^--generating system, arachidonic acid release appears to play a major role in Fc_γR-mediated O₂^- generation. In contrast, activation of C-kinase seems to be contributing mainly in the induction of both the arachidonic acid release and O₂^- generation by 12-o-tetradecanoylphorbol 13-acetate (TPA). Furthermore, suboptimal concentrations of TPA and arachidonate were found to act synergistically to stimulate O₂^- generation and the inhibition study suggested a positive synergism between C-kinase and arachidonic acid release to induce O₂^- generation. This synergistic action may have general importance in receptor-mediated O₂^- generation.