Synthesis of cysteinyl leukotrienes was assessed in patients with atopic dermatitis (AD; n = 8) and healthy volunteers (n = 8) by measuring urinary excretion of leukotriene E4 (LTE4), the main index metabolite of cysteinyl leukotrienes in man. Using this non-invasive method we demonstrated a significant (P < 0.05) 4.5-fold increase in excretion of LTE4 compared with healthy volunteers. The identity of LTE4 was unequivocally demonstrated by gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS). LTE4 was routinely measured by radioimmunoassay (RIA), and quantitative measurement of LTE4 by RIA was validated by GC-MS/MS. There was a linear correlation between LTE4 measured by RIA and by GC-MS/MS (r = 0.994). In representative samples, LTE4 was also quantitatively assessed by GC-MS/MS. In these samples, LTE4 values obtained by GC-MS/MS differed < 10% from those obtained by RIA. The present findings suggest that cysteinyl leukotrienes play a role in AD.