This study investigated the role of secretory PLA2 (sPLA2) in arachidonic acid (AA) release and lipid mediator generation in mouse bone marrow-derived mast cells. Initial studies indicated that mast cells contain multiple PLA2 activities and secreted PLA2 activity upon Ag stimulation. The secreted PLA2 activity is blocked by DTT and by a group II PLA2-neutralizing Ab. Mast cells incubated with groups I or II PLA2 selectively release polyunsaturated fatty acids, such as AA, into supernatant fluids. The bulk of AA released by sPLA2 is derived from phosphatidylethanolamine. The fatty acids released by extracellular PLA2 mimic those found in supernatant fluids after Ag stimulation of mast cells. Incubation of mast cells with PLA2 generates cyclooxygenase (CO) products but no 5-lipoxygenase (5-LO) products. Ag, but not PLA2, induces the translocation of 5-LO to cellular membranes and the formation of 5-LO products. The addition of sPLA2 to Ag-stimulated mast cells increases the synthesis of 5-LO products. Octadeuterated AA (2H8AA) added to the outside of cells to trace extracellular AA metabolism is rapidly converted to CO products. Addition of sPLA2, or Ag in combination with 2H8AA reduces the quantity of 2H8AA converted to deuterated CO products, when compared with adding 2H8AA alone. The reduction of deuterated CO products can be accounted for with increases in nondeuterated CO products. 2H8AA is only converted to 5-LO produces when mast cells are activated with Ag. The amount of 2H8AA converted to deuterated 5-LO products is reduced by the addition of PLA2 to Ag-stimulated cells. The competitive formation of deuterated and nondeuterated products observed when mast cells are incubated with 2H8AA, indicates that extracellular AA released by sPLA2 is used for both CO and 5-LO product formation. Taken together, these data reveal a role for sPLA2 in the release of AA and demonstrate that AA released by this mechanism is used for eicosanoid generation.