The airway epithelium forms a physical, chemical and immunological barrier against inhaled environmental substances. In asthma, these barrier properties are thought to be abnormal. In this study, we analysed the effect of grass pollen on the physical and immunological barrier properties of differentiated human primary bronchial epithelial cells. Following exposure to Timothy grass (Phleum pratense) pollen extract, the integrity of the physical barrier was not impaired as monitored by measuring the transepithelial resistance and immunofluorescence staining of tight junction proteins. In contrast, pollen exposure affected the immunological barrier properties by modulating vectorial mediator release. CXC chemokine ligand (CXCL)8/interleukin (IL)-8 showed the greatest increase in response to pollen exposure with preferential release to the apical compartment. Inhibition of the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways selectively blocked apical CXCL8/IL-8 release via a post-transcriptional mechanism. Apical release of CC chemokine ligand (CCL)20/macrophage inflammatory protein-3a, CCL22/monocyte-derived chemokine and tumour necrosis factor-a was significantly increased only in severe asthma cultures, while CCL11/eotaxin-1 and CXCL10/interferon-?-induced protein-10 were reduced in nonasthmatic cultures. The bronchial epithelial barrier modulates polarised release of mediators in response to pollen without direct effects on its physical barrier properties. The differential response of cells from normal and asthmatic donors suggests the potential for the bronchial epithelium to promote immune dysfunction in asthma.