A direct mass spectrometric method for 15-hydroxy metabolites of arachidonic acid and of eicosapentaenoic acid is described. 15-Hydroxyeicosanoids have the capacity to inhibit the formation of leukotrienes, potent mediators of inflammation formed by the 5-lipoxygenase pathway of arachidonic acid metabolism and may therefore have anti-inflammatory properties. 15-Hydroxyeicosatetraenoic acid (15-HETE) and 15-hydroxyeicosapentaenoic acid (15-HEPE) were synthesized using soybean lipoxygenase and arachidonic acid and eicosapentaenoic acid as substrates. These hydroxy fatty acids were then purified by reversed-phase high-performance liquid chromatography. This modified procedure provides rapid synthesis of multimilligram quantities of 15-hydroxyeicosanoids for in vitro and in vivo studies. Electron impact mass spectra of 15-HETE and 15-HEPE could be obtained directly without derivatizations.