PGE₂ is an important cyclooxygenase product that modulates airway inflammatory and smooth muscle responses. Signal transduction is mediated by four EP receptor subtypes that cause distinct effects on cell metabolism. To determine the role of EP₂ receptor activation, we produced a mouse lacking the EP₂ receptor by targeted gene disruption. The effect of aerosolized PGE₂ and other agonists was measured using barometric plethysmography and by measurements of lung resistance in mechanically ventilated mice. Inhalation of PGE₂ inhibited methacholine responses in wild-type but not in mice lacking the EP₂ receptor [EP₂(−/−)]. After airway constriction was induced by methacholine aerosol, PGE₂reduced the airway constriction enhanced pause in wild-type mice (from 0.88 ± 0.15 to 0.55 ± 0.06) but increased it in EP₂(−/−) mice (from 0.73 ± 0.08 to 1.27 ± 0.19). Similar results were obtained in mechanically ventilated mice. These data indicate that the EP₂ receptor mediates the bronchodilation effect of PGE₂.