Mechanosensitivity is hypothesized to participate in the regulation of ciliary beat frequency (CBF) in airway epithelia. To investigate this hypothesis, CBF in excised mouse trachea was monitored (microscopy image analysis) while varying mucosal shear (perfusate velocity and/or viscosity; planar flow). CBF increased within minutes of step increase to steady shear stress as small as 10⁻³ Pa and decreased within minutes of shear reduction (≤10⁻⁴ Pa). CBF response was directional, being less with cephalad vs. caudal flow, and was reduced in trachea from mutant mice lacking P2Y₂ receptors, as well as by administration of the Ca²⁺ chelator EGTA, the Ca²⁺ channel inhibitor La³⁺, the nucleotide phosphohydrolase apyrase, the metabolically stabilized adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine, the osmotic agent mannitol, and the viscosity modifier dextran. Brief exposure to exogenous ATP, a candidate mediator, augmented CBF response, although augmentation declined with higher ATP concentration (5.0 vs. 0.1 mM) or longer ATP exposure before shear (55 vs. 20 min). Prolonged extended exposure (45 min) to the metabolically stabilized ATP analog ATPγS [adenosine 5′-(3-thiotriphosphate), 0.1 mM] inhibited CBF response to shear. Furthermore, neither ATP nor ATPγS substantially increased CBF in the relative absence of shear. With viscosity increase or shear withdrawal apyrase evoked CBF stimulation, inhibitable by the adenosine receptor antagonist 8-(p-sulfophenyl)theophylline. Thus CBF response to shear is finely tuned, directional, La³⁺ sensitive, likely dependent on extracellular Ca²⁺ and ATP, involving P2Y₂ and adenosine receptor activations, influenced by shear history, tonicity, viscosity, and metabolism/exposure of ATP, and thus reflective of a complex interplay of physical and biochemical actions.