To examine the role of endogenous arachidonic acid (AA) as the possible second messenger signal in interferon-gamma (IFN-gamma) production, helper cell-depleted mouse spleen cell cultures were treated with the enzyme phospholipase A2 (PLA2). Treatment with PLA2 from several different animal sources at concentrations between 10 and 300 U/ml resulted in complete, dose-dependent restoration of competence for IFN-gamma production. By comparison, phospholipase C (PLC) from several different species failed to restore competence at concentrations between 0.3 and 30 U/ml; the inability of PLC to provide the helper signal for induction of IFN-gamma was not due to cytotoxicity. Since PLA2 provides competence for IFN-gamma production by sn-2 hydrolysis, it was of interest to identify eicosanoids and other lipids released from [3H]-AA labeled cells by PLA2 and PLC. Treatment of spleen cells with PLA2, but not PLC, resulted in the appreciable release of AA only. Sufficient AA was released from spleen cells for restoration of competence for production of IFN-gamma. All glycerol-derived cell membrane phospholipids examined (phosphatidylethanolamine, -inositol, -choline, and -serine) incorporated labeled AA which was releasable by treatment with PLA2. The data support and extend previous studies which suggested that AA plays a pivotal role in mediation of the interleukin 2 helper signal for IFN-gamma production.