The role of potential target enzymes in the protein-kinase-C-independent eicosanoid response triggered by certain bacteria in murine peritoneal macrophages [Svensson, U., Holst, E. & Sundler, R. (1991) Eur. J. Biochem. 202, 699-705] has been investigated. The eicosanoid response was found to be due to an increase in the mobilization of arachidonate rather than to inhibition of arachidonate esterification or activation of the cyclooxygenase pathway and to be accompanied by a persistent increase in the activity of the arachidonate-mobilizing phospholipase A2 (PLA2-85). Also, down-regulation of protein-kinase C by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate did not reduce the bacterial activation of PLA2-85. The increase in activity of PLA2-85, like the increase in eicosanoid formation, showed a lag period of approximately 10 min. Furthermore, exposure of 32P-labeled macrophages to either bacteria (Gardnerella vaginalis) or the protein-phosphatase inhibitor okadaic acid caused an increase in the phosphorylation of PLA2-85. Okadaic acid (0.5 microM), which itself caused arachidonate mobilization and activation of PLA2-85 after a lag period of approximately 45 min, greatly promoted the response to bacteria even at earlier time points. This study provides strong evidence that the eicosanoid response to bacteria in macrophages occurs via a protein-kinase-C-independent activation of PLA2-85 and that this activation is due to an increase in the phosphorylation of the enzyme.