Prostaglandin E(2) (PGE(2)) has complex effects on airway tone, and the existence of four PGE(2) [E-prostanoid (EP)] receptors, each with distinct signaling characteristics, has provided a possible explanation for the seemingly contradictory actions of this lipid mediator. To identify the receptors mediating the actions of PGE(2) on bronchomotor tone, we examined its effects on the airways of wild-type and EP receptor-deficient mice. In conscious mice the administration of PGE(2) increased airway responsiveness primarily through the EP1 receptor, although on certain genetic backgrounds a contribution of the EP3 receptor was detected. These effects of PGE(2) were eliminated by pretreatment with either atropine or bupivacaine and were undetectable in anesthetized mice or in denervated tracheal rings, where only EP2-mediated relaxation of airway smooth muscle was observed. Together, our findings are consistent with a model in which PGE(2) modulates airway tone by activating multiple receptors expressed on various cell populations and in which the relative contribution of these receptors might depend on the expression of modifier alleles. PGE(2)/EP1/EP3-induced airway constriction occurs indirectly through activation of neural pathways, whereas PGE(2)-induced bronchodilation results from direct activation of EP2 receptors on airway smooth muscle. This segregation of EP receptor function within the airway suggests that PGE(2) analogs that selectively activate the EP2 receptor without activating the EP1/EP3 receptors might prove useful in the treatment of asthma.