Aeroallergens produced by Alternaria alternata can elicit life-threatening exacerbations of asthma in patients sensitized to this fungus. In this study, the effect of Alternaria on ion transport mechanisms underlying mucociliary clearance and airway epithelial barrier function was investigated in human airway epithelial cells. Apical exposure to Alternaria induced an increase in anion secretion that was inhibited by blockers of CFTR and Ca²⁺-activated Cl^- channels. Stimulation of anion secretion was dependent on Ca²⁺ uptake from the apical solution. Alternaria exposure also produced an increase in reactive oxygen species (ROS) that was blocked by pretreatment with the oxidant scavenger glutathione (GSH). GSH and the NADPH oxidase inhibitor/complex 1 electron transport inhibitor diphenylene iodonium chloride (DPI) blocked ATP release and the increase in intracellular [Ca²⁺] evoked by Alternaria. Alternaria also decreased transepithelial resistance and a portion of this affect was dependent on the increase in ROS. However, the Alternaria-induced increase in unidirectional dextran (mw = 4000 Da) flux across the epithelium could not be accounted for by increased oxidative stress. These results support the conclusion that oxidative stress induced by Alternaria was responsible for regulating Ca2⁺-dependent anion secretion and tight junction electrical resistance that would be expected to affect mucociliary clearance.