Allergens are diverse proteins from mammals, birds, arthropods, plants, and fungi. Allergens associated with asthma (asthmagens) share a common protease activity that may directly impact respiratory epithelial biology and lead to symptoms of asthma. Alternaria alternata is a strong asthmagen in semiarid regions. We examined the impact of proteases from A. alternata on lung inflammation in vivo and on cleaving protease-activated receptor-2 (PAR₂) in vitro. A. alternata filtrate applied to the airway in nonsensitized Balb/c mice induced a protease-dependent lung inflammation. Moreover, A. alternata filtrate applied to human bronchial epithelial cells (16HBE14o-) induced changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), consistent with PAR₂ activation. These effects were blocked by heat inactivation or by serine protease inhibition of A. alternata filtrates, and mimicked by PAR₂ specific ligands SLIGRL-NH₂ or 2-furoyl-LIGRLO-NH₂, but not the PAR₁-specific ligand TFLLR-NH₂. Desensitization of PAR₂ in 16HBE14o- cells with 2-furoyl-LIGRLO-NH₂ or trypsin prevented A. alternata-induced [Ca²⁺]_i changes while desensitization of PAR₁, PAR₃, and PAR₄ with thrombin had no effect on A. alternata-induced Ca²⁺ responses. Furthermore, the Ca²⁺ response to A. alternata filtrates was dependent on PAR₂ expression in stably transfected HeLa cell models. These data demonstrate that A. alternata proteases act through PAR₂ to induce rapid increases in human airway epithelial [Ca²⁺]_i in vitro and cell recruitment in vivo. These responses are likely critical early steps in the development of allergic asthma.