The pathogenesis of cystic fibrosis (CF) airways disease remains controversial. Hypotheses that link mutations in CFTR and defects in ion transport to CF lung disease predict that alterations in airway surface liquid (ASL) isotonic volume, or ion composition, are critically important. ASL [Cl⁻] is pivotal in discriminating between these hypotheses, but there is no consensus on this value given the difficulty in measuring [Cl⁻] in the “thin” ASL (∼30 μm) in vivo. Consequently, a miniaturized solid-state electrode with a shallow depth of immersion was constructed to measure ASL [Cl⁻] in vivo. In initial experiments, the electrode measured [Cl⁻] in physiologic salt solutions, small volume (7.6 μl) test solutions, and in in vitro cell culture models, with ≥93% accuracy. Based on discrepancies in reported values and/or absence of data, ASL Cl⁻ measurements were made in the following airway regions and species. First, ASL [Cl⁻] was measured in normal human nasal cavity and averaged 117.3 ± 11.2 mM (n = 6). Second, ASL [Cl⁻] measured in large airway (tracheobronchial) regions were as follows: rabbit trachea and bronchus = 114.3 ± 1.8 mM; (n = 6) and 126.9 ± 1.7 mM; (n = 3), respectively; mouse trachea = 112.8 ± 4.2 mM (n = 13); and monkey bronchus = 112.3 ± 10.9 mM (n = 3). Third, Cl⁻ measurements were made in small (1–2 mm) diameter airways of the rabbit (108.3 ± 7.1 mM, n = 5) and monkey (128.5 ± 6.8 mM, n = 3). The measured [Cl⁻], in excess of 100 mM throughout all airway regions tested in multiple species, is consistent with the isotonic volume hypothesis to describe ASL physiology.