Gas chromatographic-(tandem) mass spectrometric (GC-MS(-MS)) methods for the analysis of cysteinyl leukotrienes (LTs) and leukotriene B4 (LTB4) in biological fluids were developed. The enzymatic synthesis of the stable-isotope labelled analogues [1,1-18O2] LTE4 and [14,15,17,17,18,18-2H6] LTB4 in high isotopic purity is described. Utilizing [1,1-18O2] LTE4 as an internal standard and GC-MS-MS enhanced cysteinyl LTs synthesis was found in patients with multiple trauma and psoriasis compared to healthy volunteers, respectively. The metabolism of LTB4 by human monocytes was investigated and two novel LTB4 metabolites were structurally identified by GC-MS as 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4.