Access to interstitial fluid from trachea is important for understanding tracheal microcirculation and pathophysiology. We tested whether a centrifugation method could be applied to isolate such fluid in rats by exposing excised trachea to G-forces up to 609 g. The ratio between the concentration of the equilibrated extracellular tracer ⁵¹Cr-EDTA in fluid isolated at 239 g and plasma averaged 0.94 ± 0.03 (n=14), suggesting that contamination from the intracellular fluid phase was negligible. The protein pattern of the isolated fluid resembled plasma closely and had a protein concentration 83 % of that in plasma. The colloid osmotic pressure in the centrifugate in controls (n=5) was 18.8 ± 0.6 mmHg with a corresponding pressure in plasma of 22 ± 1.5 mmHg, whereas after overhydration (n=5) these pressures fell to 9.8 ± 0.4 mmHg and 11.9 ± 0.4 mmHg, respectively. We measured inflammatory cytokine concentration in serum, interstitial and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide induced inflammation. In control animals, low levels of IL-1 , IL-6 and TNF- in serum, trachea interstitial fluid and BALF were detected. LPS resulted in a significantly higher concentration in IL-1 and IL-6 in interstitial fluid than in serum, showing a local production. To conclude, we have shown that interstitial fluid can be isolated from trachea by centrifugation and that trachea interstitial fluid has a high protein concentration and colloid osmotic pressure relative to plasma. Trachea interstitial fluid may also reflect lower airways and thus be of importance for understanding e.g. inflammatory induced airway obstruction.