Airway hyperreactivity in antigen challenged animals is mediated by eosinophil major basic protein (MBP) that blocks inhibitory M₂ muscarinic receptors on parasympathetic nerves, increasing acetylcholine release onto M₃ muscarinic receptors on airway smooth muscle. Acutely, anticholinergics block hyperreactivity in antigen challenged animals and reverse asthma exacerbations in man, but are less effective in chronic asthma. We tested whether atropine, given before antigen challenge affected hyperreactivity, M₂ receptor function, eosinophil accumulation and activation. Sensitized guinea pigs received atropine (1mg/kg, i.p.) 1 hour before challenge and 6 hours later. 24 hours after challenge, animals were anesthetized, vagotomized, paralyzed and ventilated. Airway reactivity to electrical stimulation of the vagi and to i.v. acetylcholine was not altered by atropine pretreatment in non-sensitized animals, indicating that atropine was no longer blocking post-junctional muscarinic receptors. Antigen challenge induced airway hyperreactivity to vagal stimulation that was significantly potentiated by atropine pretreatment. Bronchoconstriction induced by acetylcholine was not changed by antigen challenge or by atropine pretreatment. M₂ receptor function was lost in challenged animals, but protected by atropine pretreatment. Eosinophils in bronchoalveolar lavage and within airway tissues were significantly increased by challenge, but significantly reduced by atropine pretreatment. However, extracellular MBP in challenged airways was significantly increased by atropine pretreatment, which may account for reduced eosinophils. Depleting eosinophils with Ab IL-5 before challenge prevented hyperreactivity and significantly reduced MBP in airways of atropine pretreated animals. Thus, atropine pretreatment potentiated airway hyperreactivity by increasing eosinophil activation and degranulation. These data suggest that anticholinergics enhance eosinophil interactions with airway nerves.