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-increases to steady shear stress as small as 10^-3 Pa and decreased within minutes of shear reduction (10^-4 Pa). CBF responses were directional, being less with cephalad versus caudal flow, and were reduced in trachea from mutant mice lacking P2Y₂ receptors, as well as by administration of the [Ca²⁺]_o-chelator EGTA, the Ca²⁺-channel inhibitor La³⁺, the nucleotide phosphohydrolase apyrase, the metabolically-stabilized adenosine-receptor agonist NECA (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 this augmentation declined with higher ATP concentration (5.0 vs. 0.1 mM) or longer exposure duration before shear (55 vs. 20 min). In contrast, extended exposure (45 min, 0.1 mM) to the metabolically-stabilized ATP-analogue ATPS (adenosine 5'-(3-thiotriphosphate)) inhibited CBF response to shear. Furthermore, neither ATP nor ATPS substantially increased CBF in the relative absence of shear. Upon viscosity increase or withdrawal of shear stimulation, the presence of apyrase evoked CBF stimulation, inhibitable by the adenosine-receptor antagonist 8-SPT (8-(p-sulfophenyl)theophylline). Thus, CBF response to shear is finely tuned, directional, La³⁺-sensitive, likely [Ca²⁺]_o- and [ATP]_o-dependent, involving P2Y₂- and adenosine-receptor activations, influenced by shear history, tonicity, viscosity, and metabolism/exposure of [ATP]_o, and thus, reflective of a complex interplay of physical and biochemical actions.