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The fluid that covers the surface of conducting airways (airway surface fluid, ASF) is a critical component of one of the first defense mechanisms of the lung against microbial and other environmental insults. Despite its physiologic importance, ASF is one of the only fluids in the human body whose composition remains poorly defined and understood. Attempts to analyze ASF have been hampered greatly by the fact that it exists only as a very thin layer covering the mucosal surface of airway epithelia. To overcome some of these limitations, we have applied ultramicroanalytic techniques to microsamples collected in human airways in vivo. In contrast to previous thinking from studies on sputum samples, ASF collected from healthy airways contains much less Na and Cl (approximately 45% less) and much more K (around 600% more) than extracellular fluid or plasma (ECF), which shows that steep ion gradients exist across normal airway epithelia. These differences also show that ASF composition must be regulated and maintained by active electrolyte transport processes of airway epithelia and that it is not merely the evaporated residue of isotonic secretions or extracellular fluid exudate. However, in patients with sustained airway irritation, infection, or cystic fibrosis, we find that ASF composition appears to become more isotonic with respect to plasma and much more hypotoniv in patients with asthma.