Ozone hyperreactivity over 24 hours is mediated by blockade of inhibitory M₂ muscarinic autoreceptors by eosinophil major basic protein. Since eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to M₂ dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, M₂ function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone induced hyperreactivity to vagal stimulation persisted over three days. Although hyperreactivity one day after ozone is mediated by eosinophils, Ab VLA-4 did not inhibit either eosinophil accumulation in the lungs, around the nerves, or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs were decreased and neuronal M₂ receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from M₂ receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL, lungs and around nerves and M₂ receptors were again dysfunctional. At this point airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4 or cyclophosphamide protected M₂ function three days after ozone, and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significantly potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over three days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair.