Key points• Exposure of human bronchial epithelial (HBE) cells to fungal aeroallergens derived from Alternaria alternata stimulates Ca 2+ -dependent and Ca 2+ -independent ATP release across the apical membrane.• The Ca 2+ -dependent component was blocked by inhibitors of both ATP uptake and transport of exocytotic vesicles to the plasma membrane.• Treatment with inhibitors that target cysteine proteases significantly blocked Ca 2+ -dependent ATP release evoked by Alternaria in normal HBE cells, but not in cells derived from asthmatic patients.• The magnitude of ATP release and associated intracellular Ca 2+ mobilization was significantly greater in bronchial epithelial cells obtained from patients with asthma.• These findings establish a novel role for ATP release as a mechanism underlying Alternaria aeroallergen activation of airway mucosal immunity and that cells derived from patients with asthma exhibit greater responsiveness to these allergens.Abstract Exposure of human bronchial epithelial (HBE) cells from normal and asthmatic subjects to extracts from Alternaria alternata evoked a rapid and sustained release of ATP with greater efficacy observed in epithelial cells from asthmatic patients. Previously, Alternaria allergens were shown to produce a sustained increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that was dependent on the coordinated activation of specific purinergic receptor (P2Y 2 and P2X 7 ) subtypes. In the present study, pretreatment with a cell-permeable Ca 2+ -chelating compound (BAPTA-AM) significantly inhibited ATP release, indicating dependency on [Ca 2+ ] i . Alternaria-evoked ATP release exhibited a greater peak response and a slightly lower EC 50 value in cells obtained from asthmatic donors compared to normal control cells. Furthermore, the maximum increase in [Ca 2+ ] i resulting from Alternaria treatment was greater in cells from asthmatic patients compared to normal subjects. The vesicle transport inhibitor brefeldin A and BAPTA-AM significantly blocked Alternaria-stimulated incorporation of fluorescent lipid (FM1-43)-labelled vesicles into the plasma membrane and ATP release. In addition, inhibiting uptake of ATP into exocytotic vesicles with bafilomycin also reduced ATP release comparable to the effects of brefeldin A and BAPTA-AM. These results indicate that an important mechanism for Alternaria-induced ATP release is Ca 2+ dependent and involves exocytosis of ATP. Serine and cysteine protease inhibitors also reduced Alternaria-induced ATP release; however, the sustained increase in observed following Alternaria exposure appeared to be independent of protease-activated receptor (PAR2) stimulation.