The bronchoconstrictor response to adenosine in the actively sensitised Brown Norway rat is markedly augmented following low level allergen (ovalbumin) challenge. The response reflects activation of the A 2B receptor subtype and is mediated by 5-hydroxytryptamine (5-HT) released as a consequence of mast cell activation. We describe here the effects of wortmannin, a potent inhibitor of phosphatidylinositol-3-kinase and mast cell exocytosis, on the response to adenosine. Bronchoconstrictor responses to adenosine elicited 3 h following ovalbumin challenge were markedly and dose-dependently reduced by wortmannin given intratracheally (i.t.), 1 h prior to or 2 h post-allergen challenge. Responses to methacholine, which activates bronchial smooth muscle directly, and 5-HT were also reduced following wortmannin but to a lesser extent than those to adenosine. Bronchoconstrictor responses to adenosine 3 h post-challenge with vehicle were also markedly reduced by wortmannin given intratracheally (i.t.), 1 h prior to the “sham” challenge. Plasma histamine and 5-HT levels increased in response to adenosine given 3 h following ovalbumin challenge. The increases were suppressed by wortmannin given i.t., 2 h post-ovalbumin challenge. A reduction in the sensitivity of the airways to 5-HT explains in part the reduced bronchoconstrictor response to adenosine induced by wortmannin. A direct action to suppress 5-HT release from airway mast cells induced by adenosine also contributes to the reduction in the response. Inhibition of phosphatidylinositol-3-kinase is the presumed mechanistic basis for the observed effects.