The activation of eosinophils with the lipid mediator, leukotriene B(4), induces their homotypic aggregation. Upon activation with leukotriene B(4), eosinophils release a significant amount of arachidonic acid, a process dependent on the activation of phospholipase A(2). Here, we have evaluated whether arachidonic acid could induce aggregation of eosinophils and whether the release of arachidonic acid mediated the aggregation induced by leukotriene B(4). The exogenous administration of arachidonic acid induced a concentration-dependent eosinophil homotypic aggregation. Pretreatment of eosinophils with a 5-lipoxygenase inhibitor or a leukotriene B(4) receptor antagonist abrogated arachidonic-acid-induced aggregation. Arachidonic acid induced a significant increase in leukotriene B(4) levels and desensitised leukotriene B(4)-induced aggregation in a dose-dependent manner. Moreover, this desensitisation was effectively reversed by a 5-lipoxygenase inhibitor. However, arachidonic acid failed to induce a rise in intracellular Ca(2+) in eosinophils and failed to desensitise these cells to rises in intracellular Ca(2+) induced by leukotriene B(4). Pretreatment of eosinophils with the phospholipase A(2) inhibitor, mepacrine, inhibited the aggregation responses induced by 1 nM leukotriene B(4) by approximately 50% but had no significant effect on the other concentrations of leukotriene B(4) tested (0.1 to 100 nM). In conclusion, arachidonic acid stimulates eosinophil aggregation indirectly via the release of leukotriene B(4). Although a significant amount of arachidonic acid is released in response to activation of eosinophils with leukotriene B(4), the arachidonic acid released does appear to play a major role in mediating leukotriene B(4)-induced eosinophil aggregation.