To examine the effect of adenosine A₃ receptor stimulation on airway mucociliary clearance, we measured transport of Evans blue dye in rabbit trachea in vivo and ciliary motility of epithelium by the photoelectric method in vitro. Mucociliary transport was enhanced dose dependently by the selective A₃ agonist N⁶-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N⁶-2-(4-amino-3-iodophenyl)ethyladenosine, whereas the A₁ agonist N-cyclopentyladenosine (CPA) and the A₂ agonist CGS-21680 had no effect. The effect of IB-MECA was abolished by pretreatment with the selective A₃ antagonist MRS-1220 but not by the A₁ antagonist 1,3-dipropyly-8-cyclopentylxanthine or the A₂ antagonist 3,7-dimethyl-L-propargylxanthine. Epithelial ciliary beat frequency was increased by IB-MECA in a concentration-dependent manner, the maximal increase being 33%, and this effect was inhibited by MRS-1220. The IB-MECA-induced ciliary stimulation was not altered by the Rp diastereomer of cAMP but was greatly inhibited by Ca²⁺-free medium containing BAPTA-AM. Incubation with IB-MECA increased intracellular Ca²⁺ contents. Therefore, A₃ agonist enhances airway mucociliary clearance probably through Ca²⁺-mediated stimulation of ciliary motility of airway epithelium.