The effect of short-term coincubations of Epstein-Barr virus (EBV) with mononuclear cells on the synthesis of leukotrienes (LT) by monocytes was investigated. Although treatment of mononuclear cells with EBV alone had no significant effect on LT synthesis by monocytes, the preincubation of mononuclear cells with EBV before the further stimulation of the cells with either the ionophore A23187, the chemoattractant formyl-Met-Leu-Phe, or the phagocytic particles zymosan strikingly enhanced the formation of both LTB4 and LTC4 above the levels of synthesis observed with the stimuli alone. Such priming effect of EBV on LT synthesis was maximal after 15 minutes of preincubation of mononuclear cells with EBV and slowly declined at longer preincubation times; the priming effect of EBV was observed both in Hank's Balanced Salt Solution and plasma. The effect of EBV was abolished by prior treatment of viral particles by heat or by antibody raised against the glycoprotein gp350 of the viral envelope, but not by UV irradiation of the viral particles. Exposure of mononuclear cells to EBV was shown to strongly enhance the activation of the 5-lipoxygenase and the release of arachidonic acid induced upon cell stimulation with a second agonist. The release of arachidonic acid by the EBV-treated mononuclear cells was inhibitable by arachidonyl trifluoromethyl ketone, an inhibitor of the 80-kD cytosolic phospholipase A2. Furthermore, EBV was shown to rapidly increase (maximum effect within 15 minutes) the levels of phosphorylated form of the cytosolic phospholipase A2 (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis), a process related to the activation of this enzyme. These data show that the interaction of EBV with monocytes upregulates the formation of important lipid mediators of inflammation.