Breath analysis holds promise for non-invasive in vivo monitoring of disease related processes. However, physiological parameters may considerably affect profiles of exhaled volatile organic substances (VOCs). Volatile substances can be released via alveoli, bronchial mucosa or from the upper airways. The aim of this study was the systematic investigation of the influence of different sampling sites in the respiratory tract on VOC concentration profiles by means of a novel experimental setup. After ethical approval, breath samples were collected from 25 patients undergoing bronchoscopy for endobronchial ultrasound or bronchoscopic lung volume reduction from different sites in the airways. All patients had total intravenous anaesthesia under pressure-controlled ventilation. If necessary, respiratory parameters were adjusted to keep PETCO2 = 35–45 mm Hg. 30 ml gas were withdrawn at six sampling sites by means of gastight glass syringes: S1 = Room air, S2 = Inspiration, S3 = Endotracheal tube, S4 = Trachea, S5 = Right B6 segment, S6 = Left B6 segment (S4–S6 through the bronchoscope channel). 10 ml were used for VOC analysis, 20 ml for PCO2 determination. Samples were preconcentrated by solid-phase micro-extraction (SPME) and analysed by gas chromatography–mass spectrometry (GC-MS). PCO2 was determined in a conventional blood gas analyser. Statistically significant differences in substance concentrations for acetone, isoprene, 2-methyl-pentane and n-hexane could be observed between different sampling sites. Increasing substance concentrations were determined for acetone (15.3%), 2-methyl-pentane (11.4%) and n-hexane (19.3%) when passing from distal to proximal sampling sites. In contrast, isoprene concentrations decreased by 9.9% from proximal to more distal sampling sites. Blank bronchoscope measurements did not show any contaminations. Increased substance concentrations in the proximal respiratory tract may be explained through substance excretion from bronchial mucosa while decreased concentrations could result from absorption or reaction processes. Spatial mapping of VOC profiles can provide novel insights into substance specific exhalation kinetics and mechanisms.