Adenosine induces dyspnea, cough and airways obstruction in asthma, a phenomenon that also occurs in various sensitised animal models in which a neuronal involvement has been implicated. Whilst adenosine has been suggested to activate cholinergic nerves, the precise mechanism has not been established. In the present study, the adenosine A₁ receptor agonist, N⁶ cyclopentyl adenosine (CPA) induced a cholinergic reflex inducing tracheal smooth muscle contraction that was significantly inhibited by the adenosine A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 µg/kg) (p<0.05) in anaesthetized animals. Furthermore, the adenosine A₂ agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxoamido adenosine, CGS 21680, induced a small reflex whilst the A₃ selective agonist IB-MECA was without effect. The tracheal reflex induced by CPA was also inhibited by recurrent nerve ligation or muscarinic receptor blockade (p<0.001) indicating that a cholinergic neuronal mechanism of action accounted for this response. The cholinergic reflex in response to aerosolized CPA was significantly greater in passively sensitised compared with naive guinea pigs (p<0.01). Chronic capsaicin treatment, which inhibited sensory nerve function failed to inhibit CPA-induced reflex tracheal contractions in passively sensitised guinea pigs, although, the local anaesthetic lidocaine inhibited this response. The effects of CPA on the reflex response was not dependent upon the release of histamine from tissue mast cells or endogenous prostaglandins as shown by the lack of effect of the histamine H₁ receptor antagonist pyrilamine (1mg/kg) or the COX inhibitor meclofenamic acid (3 mg/kg), respectively. In conclusion, activation of pulmonary adenosine A₃ receptors can stimulate cholinergic reflexes and these reflexes are increased in allergic guinea-pigs.