Pseudomonas aeruginosa secrete N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule to regulate gene expression, and micromolar concentrations are found in the airway surface liquid of infected lungs. Exposure of the airway surface to C12 caused a sudden loss of transepithelial resistance (R_T) within one hour, and was characterized by a degradation and relocation of the tight junction protein ZO-1 from the apical to the basolateral pole of polarized human airway cultures (Calu-3 and primary tracheal epithelia). These effects were blocked by ZVAD-fmk, a pancaspase blocker, indicating that tight junction degradation was an early event in C12-triggered apoptosis. Short-time (10 min) pretreatment with thapsigargin (1 uM), an inhibitor of Ca²⁺ uptake into the ER, was found to be protective against the C12-induced airway epithelial barrier breakdown, and also against other apoptosis-related effects including activation of caspase 3/7 (executioner caspases in apoptosis), release of endoplasmic reticulum-targeted roGFP into the cytosol and depolarization of mitochondrial membrane potential. Pretreatment of Calu-3 airway cell monolayers with BAPTA/AM (to buffer cytosolic [Ca²⁺] (Ca_cyto) or Ca²⁺-free solutions + BAPTA/AM reduced C12-activation of apoptotic events, suggesting that C12-triggered apoptosis may involve Ca²⁺. Because C12 and thapsigargin both reduced [Ca²⁺] in the endoplasmic reticulum (Ca_er) and increased Ca_cyto, while thapsigarin increased mitochondrial [Ca²⁺] (Ca_mito) and C12 reduced Ca_mito, it is proposed that thapsigargin, may reduce C12-induced apoptosis in host cells not by raising Ca_cyto but by preventing C12-induced decreases in Ca_mito.