Aggregation of the IgE receptor on rat basophilic leukemia (RBL-2H3) cells triggers increased hydrolysis of polyphosphoinositides (PI), secretion of arachidonic acid (AA) and its metabolites, and degranulation to release 5-hydroxytryptamine. Despite the documented involvement of second messengers produced by the PI pathway in RBL cell exocytosis, recent evidence has suggested that additional signalling events are also necessary. We have, therefore, examined PLA2 activation and AA metabolite production by these cells in response to Ag stimulation, and evaluated the potential role of these in activating degranulation. The time course and antigen dose dependence for release of AA and its metabolites were comparable to those for degranulation and production of inositol phosphates (InsPs) when examined in parallel. Stimulated fatty acid release was highly selective for AA (compared with oleic or linoleic acids) and appeared to result predominantly from PLA2 activation. AA released upon antigen stimulation is rapidly metabolized to produce prostaglandin and leukotrienes. These are not required for activating degranulation, since BW755c completely inhibited AA metabolite production without affecting AA release, degranulation or InsP production. In contrast, the PLA2 inhibitors quinacrine and quercetin inhibited both AA release and degranulation in parallel, without significantly affecting levels of InsP production, and this inhibition could be partially reversed by exogenous addition of AA and lysophospholipid. These results demonstrate that activation of IgE-receptor mediated exocytosis of RBL cells does not require AA metabolites, and strongly suggest that PLA2 activation and release of AA and lysophospholipid may be involved in triggering this response.