The objective of this investigation was to determine the role of Pyk2, an intracellular non-receptor protein tyrosine kinase for post-adhesive inflammatory cell migration, on airway inflammation and hyperresponsiveness in immune sensitized mice. Blockade of Pyk2 was effected by intraperitonial administration of dominant negative C-terminal Pyk2 fused to a TAT protein transduction domain (TAT-Pyk2-CT). Ovalbumin challenge elicited infiltration of both eosinophils and lymphocytes into airways, increased mucus-containing epithelial cells, and caused increased airway hyperresponsiveness to methacholine in immune-sensitized mice. Pretreatment with 10 mg/kg TAT-Pyk2-CT i.p. blocked all of these effects and further decreased secretion of Th2 cytokine IL-4, IL-5 and IL-13 into the BAL fluid. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Pyk2-CT. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. We conclude that Pyk2, which is essential for inflammatory cell migration in vitro, regulates airway inflammation, Th2 cytokine secretion and airway hyperresponsiveness in the ovalbumin-sensitized mice during antigen challenge in vivo.