BACKGROUND The diagnostic potential of breath analysis has been limited by a lack of knowledge on origin, distribution, and metabolism of the exhaled substances. To overcome this problem, we developed a method to assess trace amounts of hydrocarbons (pentane and isoprene), ketones (acetone), halogenated compounds (isoflurane), and thioethers (dimethyl sulfide) in the blood of humans and animals. METHODS Arterial and venous blood samples were taken from mechanically ventilated patients. Additional blood samples were taken from selected vascular compartments of 19 mechanically ventilated pigs. Volatile substances were concentrated by means of solid-phase microextraction (SPME), separated by gas chromatography, and identified by mass spectrometry. RESULTS Detection limits were 0.02-0.10 nmol/L. Venous concentrations in pigs were 0.2-1.3 nmol/L for isoprene, 0-0.3 nmol/L for pentane, and 1.2-15.1 nmol/L for dimethyl sulfide. In pigs, substances were not equally distributed among vascular compartments. In humans, median arteriovenous concentration differences were 3.58 nmol/L for isoprene and 1.56 nmol/L for pentane. These values were comparable to pulmonary excretion rates reported in the literature. Acute respiratory distress syndrome (ARDS) patients had lower isoprene concentration differences than patients without ARDS. CONCLUSIONS The SPME method can detect volatile substances in very low concentrations in the blood of humans and animals. Analysis of volatile substances in vascular compartments will enlarge the diagnostic potential of breath analysis.