A standard procedure for exhaled breath condensate (EBC) collection is still lacking. The aim of this study was to compare the concentration of several biomarkers in whole (W-EBC) and fractionated EBC (A-EBC), the latter collected starting from CO2 ≥ 50% increase during exhalation. Forty-five healthy non-smokers or asymptomatic light smokers were enrolled. Total protein concentrations in W-EBC and A-EBC were overlapping (median: 0.7 mg l(-1) in both cases), whereas mitochondrial DNA was higher in A-EBC (0.021 versus 0.011 ng ml(-1)), indicating a concentration rather than a dilution of lining fluid droplets in the last portion of exhaled air. H2O2 (0.13 versus 0.08 µM), 8-isoprostane (4.9 versus 4.4 pg ml(-1)), malondialdehyde (MDA) (4.2 versus 3.2 nM) and 4-hydroxy-2-nonhenal (HNE) (0.78 versus 0.66 nM) were all higher in W-EBC, suggesting a contribution from the upper airways to oxidative stress biomarkers in apparently healthy subjects. NH4(+) was also higher in W-EBC (median: 590 versus 370 µM), with an estimated increase over alveolar and bronchial air by a factor 1.5. pH was marginally, but significantly higher in W-EBC (8.05 versus 8.01). In conclusion, the fractionation of exhaled air may be promising in clinical and occupational medicine.