Arachidonic acid is released by phospholipase A(2) and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and systematic investigation of these biological processes requires the simultaneous quantitation of a large number of eicosanoids in a single analysis. We have developed a robust liquid chromatography/tandem mass spectrometry method that can identify and quantitate over 60 different eicosanoids in a single analysis, and we applied it to agonist-stimulated RAW264.7 murine macrophages. Fifteen different eicosanoids produced through COX and 5-LO were detected either intracellularly or in the media following stimulation with 16 different agonists of Toll-like receptors (TLR), G protein-coupled receptors, and purinergic receptors. No significant differences in the COX metabolite profiles were detected using the different agonists; however, we determined that only agonists creating a sustained Ca(2+) influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca(2+) and TLR pathways was detected and discovered to be independent of NF-kappaB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A(2)-mediated eicosanoid production work through two distinct pathways.