Muscarinic cholinergic signaling plays an essential role in the control of the normal airway functions and in the development of pulmonary pathologies including asthma. In this paper we demonstrate that the airways of mice deficient in a cAMP-specific phosphodiesterase (PDE4D) are no longer responsive to cholinergic stimulation. Airway hyperreactivity that follows exposure to antigen was also abolished in PDE4D^−/− mice, despite an apparently normal lung inflammatory infiltration. The loss of cholinergic responsiveness was specific to the airway, not observed in the heart, and was associated with a loss of signaling through muscarinic receptors with an inability to decrease cAMP accumulation. These findings demonstrate that the PDE4D gene plays an essential role in cAMP homeostasis and cholinergic stimulation of the airway, and in the development of hyperreactivity. In view of the therapeutic potentials of PDE4 inhibitors, our findings provide the rationale for novel strategies that target a single PDE isoenzyme.