Control of airway smooth muscle is provided by parasympathetic nerves that release acetylcholine onto M₃ muscarinic receptors. Acetylcholine release is limited by inhibitory M₂ muscarinic receptors. In antigen-challenged guinea pigs, hyperresponsiveness is due to blockade of neuronal M₂ receptors by eosinophil major basic protein (MBP). Because exposure of guinea pigs to ozone also causes M₂dysfunction and airway hyperresponsiveness, the role of eosinophils in ozone-induced hyperresponsiveness was tested. Animals were exposed to filtered air or to 2 parts/million ozone for 4 h. Twenty-four hours later, the muscarinic agonist pilocarpine no longer inhibited vagally induced bronchoconstriction in ozone-exposed animals, indicating M₂ dysfunction. M₂ receptor function in ozone-exposed animals was protected by depletion of eosinophils with antibody to interleukin-5 and by pretreatment with antibody to guinea pig MBP. M₂ function was acutely restored by removal of MBP with heparin. Ozone-induced hyperreactivity was also prevented by antibody to MBP and was reversed by heparin. These data show that loss of neuronal M₂ receptor function after ozone is due to release of eosinophil MBP.