Cysteinyl leukotrienes (CysLTs) can be measured in exhaled breath condensate (EBC); however, there is considerable variation in reported EBC CysLT concentrations from asthmatic and healthy subjects between published studies, which may be partially explained by CysLT degradation during processing and storage. We assessed CysLT stability over 6 months in EBC from healthy subjects stored at -80 °C, layered with argon and then stored at -80 °C or stored in 0.2% formic acid in methanol at -80 °C following solid-phase extraction (SPE). We found significant CysLT degradation over time in both spiked and unspiked EBC samples stored at -80 °C or layered with argon. CysLT recovery was significantly greater after storage for 6 months in 0.2% formic acid in methanol following SPE; however, there was substantial variability in endogenous CysLT recovery over time, possibly attributable to inter- and intra-assay variability at the low end of the CysLT assay range. Despite the greater recovery of CysLTs in EBC stored in methanol following SPE, the degree of variability introduced by this method appears unacceptably high. We believe that the development of more sensitive and less variable methods for quantifying CysLTs in EBC are required before CysLTs can reliably be utilized as biomarkers in exhaled breath. Sample processing and storage, as well as inter- and intra-assay variability, should be carefully considered in the design of clinical studies that include assessments of EBC constituents as biomarkers.