Background Group 1 allergens from grass pollen (e.g. Phl p 1, the major allergen of timothy grass Phleum pratense) cause IgE reactivity in about 95% of allergic subjects and exist in all grass species. The respiratory epithelium represents a first line of contact of the immune system with airborne allergens, functions as physical barrier and is an important immunological regulation system. Objective The aim of this study was to investigate the interaction of Phl p 1 with human respiratory epithelium to elucidate the contribution of epithelial cells to the development of allergic reactions. Methods Purified Phl p 1 was used to stimulate A549 cells and transient transfected HEK293 cells. mRNA level of different mediators were investigated by real-time PCR, release of the mediators was determined by ELISA. The 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) test and an ex vivo model of the murine trachea were used to investigate a potential proteolytic activity of Phl p 1. Results Phl p 1 activates respiratory epithelial cells as measured by induction of IL-6, IL-8 and TGF-β mRNA and release. Phl p 1, in contrast to Der p 1 from the house dust mite, does not exert proteolytic activity, as investigated by microscopic observation and MTT test. In an ex vivo model of the murine trachea we were able to show that Der p 1, in contrast to Phl p 1, enhances the transportation velocity of particles by the trachea, presumably by ATP released from the injured epithelium. Conclusion We conclude that under physiological conditions Phl p 1 affects tracheal epithelial cells through a non-proteolytic activity. Enhancement of TGF-β expression induced by Phl p 1 together with the increased release of IL-6 and IL-8 might provide an indirect mechanism through which the allergen may cross the epithelial barrier and attracts immunocompetent cells.