Extracellular nucleotides are involved in the control of mucociliary clearance (MCC) in human airways. Because of their therapeutic potential for obstructive lung diseases such as cystic fibrosis (CF), we investigated nucleotide metabolism on airway epithelial surfaces. The mucosal surface of human nasal and bronchial epithelial cells in culture dephosphorylated ATP into ADP, AMP, and adenosine. Bronchial cells hydrolyzed ATP at a faster rate than nasal cells, 3.8 ± 0.5 nmol·min^–1 cm^–2 and 2.2 ± 0.4 nmol·min^–1 cm^–2, respectively. The ratios of ATP/ADP hydrolysis for bronchial and nasal epithelia were 2.9 ± 0.6 and 2.1 ± 0.4, respectively. Divalent cations were required for ATP hydrolysis (Ca²⁺ > Mg²⁺) and pH dependency profile revealed two peaks, at pH 7.5 ad 9.0. Kinetic analysis supported the coexpression of more than one ATP‐hydrolyzing activity on nasal (K_m = 17 ± 2 μM, 129 ± 5 μM, and 405 ± 12 μM) and bronchial (K_m = 12 ± 5 μM and 136 ± 8 μM) epithelial cells. Reverse‐transcriptase polymerase chain reaction confirmed the coexpression of all three families of ATP‐hydrolyzing ectoenzymes: ecto‐nucleoside triphosphate diphosphohydrolases, ecto‐nucleotide pyrophosphatase/phosphodiesterases, and alkaline phosphatases. Altogether, these findings demonstrate the presence of a multienzyme system in human airways, with the capacity to control ATP concentrations under physiological and pathological conditions. The identification of the major enzyme(s) responsible for ATP hydrolysis in human airways may lead to the development of specific inhibitors that would improve nucleotide‐based treatments of MCC in patients with CF. Drug Dev. Res. 52:66–75, 2001. © 2001 Wiley‐Liss, Inc.