Background: Staphylococcus aureus enterotoxin B (SEB) has recently been postulated to be involved in the pathology of granulocyte-dominated disease. Studying the immunologic interaction between SEB and airway epithelial cells in immortalized cell lines or long-term epithelial cell cultures has obvious disadvantages. Methods: We used a novel technique of freshly isolated and purified human nasal epithelial cells (HNEC) from healthy, nonallergic individuals, which were incubated for 24 h without/with SEB at different concentrations. Chemokine production was evaluated in the supernatant using Cytometric Bead Array. The chemotactic activity of the supernatant was studied in vitro using a Boyden chamber. Survival was evaluated with flow cytometry, using propidium iodide to identify dead cells. Results: Staphylococcus aureus enterotoxin B showed a dose-dependent induction of interferon-inducible protein-10, monokine induced by interferon-γ, regulated upon activation normal T cell expressed and secreted, monocyte chemoattractant protein 1 (MCP-1) and granulocyte colony stimulating factor production by epithelial cells in vitro. The supernatant of epithelial cells had chemotactic activity for granulocytes in vitro, which was enhanced in the supernatant of SEB-stimulated epithelial cells. Reduced number of propidium iodide positive granulocytes was found in the conditions where supernatant of SEB-stimulated epithelial cells was applied. Conclusion: Staphylococcus aureus enterotoxin B exerts a direct pro-inflammatory effect on HNEC, with induction of chemokine and growth factor release, resulting in the migration and prolonged survival of granulocytes in vitro.