Background Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. Objective We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. Methods We cultured the human bronchial epithelial cell line 16HBE14o− in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125–labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. Results Culture supernatants from activated allergen-specific T H1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-γ, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-α) identified IFN-γ as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-γ–mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. Conclusion These results demonstrate that the T H1 cell–derived cytokine IFN-γ facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.