Abstract-Airway inflammation characterizing asthma and other airway diseases may be monitored through biomarker analysis of exhaled breath condensate (EBC). In an attempt to discover novel EBC biomarkers, a high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was used to analyze EBC from ten control non-asthmatics and one asthmatic individual for five fatty acid metabolites: 9,12,13-trihydroxyoctadecenoic acid (9,12,13-TriHOME), 9,10,13-TriHOME, 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME), 12-hydroxyeicosatetraenoic acid (12-HETE), and 12(13)-epoxyoctadecenoic acid (12(13)-EpOME). The method was shown to be sensitive, with an on-column limit of quatitation (LOQ) in the pg range (corresponding to pM concentrations in EBC), and linear over several orders of magnitude for each analyte in the calibrated range. Analysis of EBC spiked with the five fatty acid metabolites was within 81%-119% with only a few exceptions. Endogenous levels in EBC exhibited intraand inter-assay precision of 10%-22%, and 12%-36%, respectively. EBC from the healthy subjects contained average analyte levels between 15 and 180 pM with 12-HETE present above the LOQ in only one of the subjects at a concentration of 240 pM. Exposure of the asthmatic subject to allergen led to increased EBC concentrations of 9,12,13-TriHOME, 9,10,13-TriHOME, 12,13-DiHOME, and 12(13)-EpOME when compared to levels in EBC collected prior to allergen exposure (range =40-510 pM). 12,13-DiHOME was significantly increased (Student's t-test, p < 0.05). In conclusion, we have developed a new HPLC-ESI-MS/MS method for the analysis of five fatty acid metabolites in EBC, which are potential biomarkers for asthma monitoring and diagnosis.