Urocortins are members of the corticotropin-releasing factor (CRF) family of peptides that bind to two receptors, CRF₁ and CRF₂. While CRF₁ is a high-affinity receptor for CRF, urocortin III binds with much greater affinity to CRF₂. In the present study we investigated the effect of CRF₂ receptor activation with urocortin III on airway smooth muscle tone in vitro and in an acute model of airway inflammation in mice. Urocortin III caused relaxation of methacholine-precontracted mouse tracheal segments. CRF caused similar relaxation, but with reduced potency compared to urocortin III, consistent with the CRF₂ receptor subtype. Relaxation induced by urocortin III was concentration-dependently inhibited by the CRF₂ antagonist, astressin 2B, with an IC₅₀ in the nanomolar range. These relaxations were potentiated by inhibition of phosphodiesterases but unaffected by inhibition of cyclooxygenase and NO or by removal of the epithelium. Finally, the number of neutrophils retrieved by bronchoalveolar lavage after administration of bacterial LPS (LPS) was reduced by prior intraperitoneal (i.p.) injection of urocortin III. This effect was also suppressed by astressin 2B, implicating CRF₂ receptors. Therefore, CRF₂ agonists appear to have both bronchorelaxant and anti-inflammatory activities and might represent an interesting therapeutic approach to the treatment of inflammatory lung diseases.--Moffatt, J. D., Lever, R., Page, C. P. Activation of corticotropin-releasing factor receptor-2 (CRF₂) causes bronchorelaxation and inhibits pulmonary inflammation in mice.